Methods for the treatment of a traumatic central nervous system injury

ABSTRACT

Methods of treating a subject with a traumatic central nervous system injury, more particularly, a traumatic brain injury, are provided. The methods comprise a therapy comprising a constant or a two-level dosing regime of progesterone. In one method, a subject in need thereof is administered at least one cycle of therapy, wherein the cycle of therapy comprises administering a therapeutically effective two-level intravenous dosing regime of progesterone. The two-level dosing regime comprises a first time period, wherein a higher hourly dose of progesterone is administered to the subject, followed by a second time period, wherein a lower hourly dose of progesterone is administered to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/527,816, filed Sep. 27, 2006, which claims the benefit ofInternational Patent Application No. PCT/US2006/010797, filed Mar. 24,2006, U.S. Provisional Patent Application No. 60/664,728, filed Mar. 24,2005, and U.S. Provisional Application No. 60/729,663, filed Oct. 24,2005; each of which is hereby incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support under 1R01N5 39097-01A1 awarded by the National Institute of NeurologicalDisorders and Stroke (NINDS), National Institutes of Health. The UnitedStates Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to methods for treating a traumatic injury to thecentral nervous system.

BACKGROUND OF THE INVENTION

Between 1.5 and 2 million Americans sustain a traumatic brain injury(TBI) each year (Anonymous, “Traumatic Brain Injury,” Center for DiseaseControl and Prevention, National Center for Injury Prevention andControl, 2003, Vol. 2003). In the U.S. it is estimated that TBI isresponsible for 50,000 deaths and 100,000 hospitalizations annually(Anonymous, “Traumatic Brain Injury,” Center for Disease Control andPrevention, National Center for Injury Prevention and Control, 2003,Vol. 2003). Over 80,000 are disabled annually, approximately 17,000 ofwhom require specialized care for life (Kraus (1997) “Epidemiology ofHead Injury,” in Head Injury, ed. Cooper (Williams & Wilkins Co.,Baltimore) pp 1-19; Selecki et al. (1982) Australian & New ZealandJournal of Surgery 52(1):93-102). In addition to the initial lesioncreated by abrupt trauma to the brain, excessive biomechanical forceinitiates a cascade of secondary deleterious events that candramatically increase lesion size, morbidity, and mortality for days tomonths after the initial injury (McIntosh et al. (1996) Lab Invest,74(2):315-42; Stambrook et al. (1990) Can J Surg 33(2): 115-8). Despitethe enormity of the problem, an effective pharmacological treatment forTBI in humans has not been identified.

Continuous intravenous (IV) infusion allows rapid drug delivery andachievement of a continuous steady state serum concentration, but thisroute for administration of progesterone is not FDA approved in theUnited States. Only three human studies involving the use of IVprogesterone in the US have been reported. In an FDA-approved (IND33,580) phase I clinical trial, Christen, et al. administered IVprogesterone dissolved in an ethanol-Intralipid 20% fat emulsioncombined with doxorubicin over 24 hours to 32 cancer patients withouttoxic effects (Christen et al. (1993) Journal of Clinical Oncology11(12):2417-2426). In a second study, Allolio et al. reported thatsteady state serum concentrations (C_(SS)) of progesterone could beachieved in healthy male volunteers (Allolio et al. (1995) EuropeanJournal of Endocrinology 133(6):696-700). The third study was modeledafter the study performed by Christen et al, but was a phase II trialtesting the effect of coadministration of high-dose progesterone on thepharmacokinetics of paclitaxel. The manuscript did not present detailedinformation on the pharmacokinetics of progesterone.

Following a traumatic injury to the central nervous system, a cascade ofphysiological events leads to neuronal loss including, for example, aninflammatory immune response and excitotoxicity resulting from theinitial impact disrupting the glutamate, acetylcholine, cholinergic,GABA_(A), and NMDA receptor systems. In addition, the traumatic CNSinjury is frequently followed by brain and/or spinal cord edema thatenhances the cascade of injury and leads to further secondary cell deathand increased patient mortality. Methods are needed for the in vivotreatment of traumatic CNS injuries that are successful at providingsubsequent trophic support to remaining central nervous system tissue,and thus enhancing functional repair and recovery, under the complexphysiological cascade of events which follow the initial insult.

SUMMARY OF THE INVENTION

Methods of treating a subject with a traumatic central nervous systeminjury, more particularly, a traumatic brain injury, are provided. Themethods comprise treatment of a traumatic brain injury in a humansubject by administering to the subject in need thereof atherapeutically effective concentration of progesterone or syntheticprogestin. In specific methods, treatment of a traumatic brain injury ina human subject comprises a therapy comprising a constant or a two-leveldosing regime of progesterone or synthetic progestin. In furthermethods, the constant or two-level dosing regime of progesterone orsynthetic progestin results in a serum progesterone or syntheticprogestin level of about 100 ng/ml to about 1000 ng/ml. In othermethods, the constant or two-level dosing regime results in a serumprogesterone or synthetic progestin level of less than 450 ng/ml.

Further provided is a method of treating a traumatic brain injury in ahuman subject. The method comprises administering to the subject in needthereof at least one cycle of therapy, wherein the cycle of therapycomprises administering a therapeutically effective two-levelintravenous dosing regime of progesterone or synthetic progestin. Thetwo-level dosing regime can comprise a first time period, wherein ahigher hourly dose of progesterone or synthetic progestin isadministered to the subject, followed by a second time period, wherein alower hourly dose of progesterone or synthetic progestin is administeredto the subject. In specific methods, the first time period comprises anhourly dose of progesterone or synthetic progestin of about 0.1 mg/kg toabout 7 mg/kg. In other methods, the second time period comprises anhourly dose of progesterone or synthetic progestin of about 0.05 mg/kgto about 5 mg/kg. In other methods, a third time period comprising atapered administration protocol is added to the progesterone orsynthetic progestin dosing regime.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows stable progesterone concentrations can be achieved rapidlyusing continuous intravenous infusion. The closed circles represent theserum concentration-time profile for one patient receiving progesterone.The solid triangles represent the serum concentration-time profile for apatient receiving a placebo infusion. Progesterone concentrations forpatients receiving a placebo infusion remained constant throughout thestudy period. C_(SS) concentrations in patients receiving progesteroneare rapidly reached and, once achieved, are stable throughout theinfusion period.

FIG. 2 shows there is a significant correlation between predicted andmeasured C_(SS). C_(SS) were predicted as the ratio of infusion rate andCL. The predicted values were compared to C_(SS) measured for eachpatient by plotting each pair of values against the line of identity.The Spearman Rank correlation coefficient for this relationship was0.946 (p<0.001).

FIG. 3 shows bland-Altman analysis of the correlation between predictedand measured C_(SS). Because a plot of predicted versus measured C_(SS)often do not reveal a systematic under or over estimation (bias), aBland-Altman analysis was conducted. The averages of the measured andpredicted values (abcissa) are plotted against the relative differencein the two values (ordinate). The solid line is the mean value for therelative difference (−0.8±12.2%; mean±SD) and the dotted lines representthe 95% confidence intervals for the data. This plot clearlydemonstrates that there is no significant bias associated with thismethod of prediction.

FIG. 4 shows C_(SS) values a consistently lower than those predictedbased on previously reported pharmacokinetic parameters. Measured C_(SS)for the 21 males (solid circles) and 11 females (solid triangles) areindividually plotted. The solid and dotted lines represent our originaltarget concentrations of 450±100 ng/mL. These data clearly demonstratethat in TBI patients, C_(SS) values are significantly lower thanpredicted using pharmacokinetic parameters previously reported.

FIG. 5 provides a schematic of the enrollment protocol for the selectionof patient for the TBI study.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention relates to methods of treating a human subjectwith a traumatic central nervous system injury, more particularly, atraumatic brain injury. The methods comprise treatment of a traumaticbrain injury in a human subject by administering to the subject in needthereof a therapeutically effective concentration of progesterone orsynthetic progestin. As discussed in more detail below, the methods fortreating a traumatic brain injury in a human subject comprise a therapycomprising a dosing regime of progesterone or synthetic progestin.

A traumatic injury to the CNS is characterized by a physical impact tothe central nervous system. For example, a traumatic brain injuryresults when the brain is subjected to a physical force that results inprogressive neuronal cell damage and/or cell death. A traumatic braininjury may result from a blow to the head and manifest as either an openor closed injury. Severe brain damage can occur from lacerations, skullfractures, and conversely, even in the absence of external signs of headinjury. Accordingly, the methods of the invention can be used to treat atraumatic brain injury, including, blunt traumas, as well as,penetrating traumas.

The physical forces resulting in a traumatic brain injury may causetheir effects by inducing three types of injury: skull fracture,parenchymal injury, and vascular injury. Parenchymal injuries includeconcussion, direct parenchymal injury and diffuse axonal injury.Concussions are characterized as a clinical syndrome of alteration ofconsciousness secondary to head injury typically resulting from a changein the momentum of the head (movement of the head arrested against aridged surface). The pathogenesis of sudden disruption of nervousactivity is unknown, but the biochemical and physiological abnormalitiesthat occur include, for example, depolarization due to excitatory aminoacid-mediated ionic fluxes across cell membranes, depletion ofmitochondrial adenosine triphosphate, and alteration in vascularpermeability. Postconcussive syndrome may show evidence of directparenchymal injury, but in some cases there is no evidence of damage.

Contusion and lacerations are conditions in which direct parenchymalinjury of the brain has occurred, either through transmission of kineticenergy to the brain and bruising analogous to what is seen in softtissue (contusion) or by penetration of an object and tearing of tissue(laceration). A blow to the surface of the brain leads to rapid tissuedisplacement, disruption of vascular channels, and subsequenthemorrhage, tissue injury and edema. Morphological evidence of injury inthe neuronal cell body includes pyknosis of nucleus, eosinophilia of thecytoplasm, and disintegration of the cell. Furthermore, axonal swellingcan develop in the vicinity of damage neurons and also at greatdistances away from the site of impact. The inflammatory response to theinjured tissue follows its usual course with neutrophiles preceding theappearance of macrophages.

In accordance with the methods of the present invention, progesterone orsynthetic progestin is used to promote a positive therapeutic responsewith respect to the traumatic central nervous system injury. By“treatment” is intended any improvement in the subject having thetraumatic CNS injury including both improved morphological recovery(i.e., enhanced tissue viability) and/or behavioral recovery. Theimprovement can be characterized as an increase in either the rateand/or the extent of behavioral and anatomical recovery following thetraumatic CNS injury. Accordingly, a “positive therapeutic response”includes both a complete response and a partial response. Variousmethods to determine if a complete or a partial therapeutic response hasoccurred are discussed in detail elsewhere herein.

Neurodegeneration is the progressive loss of neurons in the centralnervous system. As used herein, “neuroprotection” is the arrest and/orreverse progression of neurodegeneration following a traumatic centralnervous system injury. Multiple physiological events lead to theneurodegeneration of the CNS tissues following a traumatic CNS injury.These events include, for example, cerebral edema, destruction ofvascular integrity, increase in the immune and inflammatory response,demyelinization, and lipid peroxidation. Hence, the methods of theinvention also find use in reducing and/or preventing the physiologicalevents leading to neurodegeneration. Specifically, the present inventionprovides methods for reducing or eliminating neuronal cell death, edema,ischemia, and enhancing tissue viability following a traumatic injury tothe central nervous system.

The progesterone or synthetic progestin therapy of the invention isadministered to a subject having a traumatic CNS injury. As definedherein, the subject can be any mammal, preferably a human. In specificembodiments, the human is an adult (over 18 years of age), while inother embodiments, the human is a child (under 18 years of age). Thechild can be a neonate, infant, toddler, pre-pubescent or post-pubescentand range in age from about birth, 1 month to about 2 year, about 1 yearto about 5 years, about 4 years to about 9 years, about 8 years to about14, or about 13 to about 18 years of age. In addition, the human can beabout 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to90, 90 to 95 or older.

The present invention provides a method of treating a traumatic CNSinjury by administering to a subject progesterone or synthetic progestinin a therapeutically effective amount. By “therapeutically effectiveamount” is meant the concentration of a progesterone or syntheticprogestin that is sufficient to elicit a therapeutic effect. Thus, theconcentration of a progesterone or synthetic progestin in anadministered dose unit in accordance with the present invention iseffective in the treatment or prevention of neuronal damage that followsa traumatic injury to the CNS and hence, elicits a neuroprotectiveeffect. The therapeutically effective amount will depend on many factorsincluding, for example, the specific activity of the progesterone orsynthetic progestin, the severity and pattern of the traumatic injury,the resulting neuronal damage, the responsiveness of the patient, theweight of the patient, along with other intraperson variability, themethod of administration, and the progesterone or synthetic progestinformulation used.

The compositions comprising the therapeutically effective concentrationof progesterone or synthetic progestin may be administered using anyacceptable method known in the art. Thus, for example, thepharmaceutical composition comprising progesterone or syntheticprogestin can be administered by any method, including intravenous (IV)injection, intramuscular (IM) injection, subcutaneous (SC) injection, orvaginal administration. In specific embodiments of the invention, thepharmaceutical composition comprising progesterone or syntheticprogestin is administered by IV injection. When administeredintravenously, the pharmaceutical composition comprising theprogesterone or synthetic progestin can be administered by infusion overa period of about 1 to about 120 hours. In some embodiments, infusion ofthe progesterone or synthetic progestin occurs over a period of about 24to about 72 hours, over a period of about 48 to about 96 hours, or overa period of about 24 to about 120 hours.

In one embodiment of the present invention, progesterone or syntheticprogestin is administered via parenteral (including intraperitoneal,intravenous, subcutaneous, or intramuscular) administration in a dose ofabout 0.1 ng to about 100 g per kg of body weight, about 10 ng to about50 g per kg of body weight, from about 100 ng to about 1 g per kg ofbody weight, from about 1 μg to about 100 mg per kg of body weight, fromabout 1 μg to about 50 mg per kg of body weight, from about 0.01 mg toabout 10 mg per kg of body weight, from about 0.05 mg to about 5 mg perkg of body weight, from about 0.1 mg to about 1 mg per kg of bodyweight, from about 0.1 mg to about 0.5 mg per kg of body weight, fromabout 0.5 mg to about 1 mg per kg of body weight, from about 0.5 mg toabout 0.7 mg per kg of body weight, from about 0.7 mg to about 1 mg perkg of body weight, from about 0.1 mg to about 7 mg per kg of bodyweight, from about 0.1 mg to about 7.1 mg per kg of body weight, fromabout 0.4 to about 0.6 mg/kg, from about 0.45 to about 0.55 mg/kg, about0.5 mg/kg, from about 0.6 to about 0.8 mg/kg, from about 0.65 to about0.75 mg/kg, about 0.7 mg/kg, from about 1 mg to about 500 mg per kg ofbody weight; and from about 1 mg to about 50 mg per kg of body weight.Alternatively, the amount of progesterone or synthetic progestinadministered to achieve a therapeutic effective dose is about or atleast about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 0.2 mg,0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight orgreater.

In one embodiment of the present invention, progesterone or syntheticprogestin is administered via a constant dosing regimen in a dose ofabout 0.01 to about 10 mg/kg/h, from about 0.05 to about 5 mg/kg/h, fromabout 0.1 to about 1 mg/kg/h, from about 0.4 to about 0.6 mg/kg/h, fromabout 0.45 to about 0.55 mg/kg/h, about 0.5 mg/kg/h, from about 0.6 toabout 0.8 mg/kg/h, from about 0.65 to about 0.75 mg/kg/h, or about 0.7mg/kg/h. Alternatively, the amount of progesterone or syntheticprogestin administered to achieve a therapeutic effective dose is aboutor at least about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 0.2mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg,3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight per houror greater.

Progesterone or synthetic progestin may be administered once or severaltimes a day. The duration of the treatment may be once per day for aperiod of about 1, 2, 3, 4, 5, 6, 7 days or more. The daily dose can beadministered either by a single dose in the form of an individual dosageunit or several smaller dosage units or by multiple administration ofsubdivided dosages at certain intervals.

For instance, a dosage unit can be administered from about 0 hours toabout 1 hr, about 1 hr to about 24 hr, about 1 to about 72 hours, about1 to about 120 hours, or about 24 hours to at least about 120 hours postinjury. Alternatively, the dosage unit can be administered from about0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 30, 40, 48, 72, 96, 120 hours or longer post injury.Subsequent dosage units can be administered any time following theinitial administration such that a therapeutic effect is achieved. Forinstance, additional dosage units can be administered to protect thesubject from the secondary wave of edema that may occur over the firstseveral days post-injury.

In specific embodiments of the invention, the subject undergoing thetherapy with progesterone or synthetic progestin is administered aconstant progesterone or synthetic progestin dosing regimen. By“constant progesterone or synthetic progestin dosing regimen” isintended the subject undergoing therapy with progesterone or syntheticprogestin is administered a constant total hourly infusion dose ofprogesterone or synthetic progestin over the course of treatment. Thishourly dose of progesterone or synthetic progestin is partitioned into aseries of equivalent doses that are administered according to anappropriate dosing schedule depending on the method of administration.The duration of the constant progesterone or synthetic progestin dosingregimen is about 12, 24, 36, 60, 72, 84, or 120 hours or about 1 to 24hours, about 12 to 36 hours, about 24 to 48 hours, about 36 to 60 hours,about 48 to 72 hours, about 60 to 96 hours, about 72 to 108 hours, about96 to 120 hours, or about 108 to 136 hours.

In other embodiments of the invention, the therapy with the progesteroneor synthetic progestin comprises a “two-level progesterone or syntheticprogestin dosing regimen.” By “two-level progesterone or syntheticprogestin dosing regimen” is intended the subject undergoing the therapywith progesterone or synthetic progestin is administered progesterone orsynthetic progestin during two time periods of progesterone or syntheticprogestin dosing. The two-time periods can have a combined duration ofabout 12 hours to about 7 days, including, 1, 2, 3, 4, or 5 days orabout 15, 15, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, or 144 hours or about 1 to24 hours, about 12 to 36 hours, about 24 to 48 hours, about 36 to 60hours, about 48 to 72 hours, about 60 to 96 hours, about 72 to 108hours, about 96 to 120 hours, or about 108 to 136 hours. In oneembodiment, the two-level progesterone or synthetic progestin dosingregimen has a combined duration of about 1 day to about 5 days; in otherembodiments, the two-level progesterone or synthetic progestin dosingregimen has a combined duration of about 1 day to about 3 days.

In one embodiment, the total hourly dose of progesterone or syntheticprogestin that is to be administered during the first and second timeperiods of the two-level progesterone or synthetic progestin dosingregimen is chosen such that a higher total infusion dose of progesteroneor synthetic progestin per hour is given during the first time periodand a lower infusion dose of progesterone or synthetic progestin perhour is given during the second time period. The duration of theindividual first and second time periods of the two-level progesteroneor synthetic progestin dosing regimen can vary, depending upon thehealth of the individual and history of the traumatic injury. Generally,the subject is administered higher total infusion dose of progesteroneor synthetic progestin per hour for at least 1, 2, 3, 4, 5, 6, 12 or 24hours out of the 1 day to 5 day two-level progesterone or syntheticprogestin dosing regimen. The length of the second time period can beadjusted accordingly, and range for example, from about 12 hrs, 24 hrs,36 hrs, 48 hrs, 60 hrs, 72 hrs, 84 hrs, 96 hrs, 108 hrs, 120 hrs orabout 12 to about 36 hrs, about 24 to about 36 hrs, about 24 to about 48hrs, about 36 hrs to about 60 hours, about 48 hrs to about 72 hrs, about60 hrs to about 84 hours, about 72 hrs to about 96 hrs, or about 108 hrsto about 120 hrs. Thus, for example, where the two-level progesterone orsynthetic progestin dosing regimen has a combined duration of 3 days,the higher total doses of progesterone or synthetic progestin could beadministered for the first hour, and the lower total hourly dose ofprogesterone or synthetic progestin could be administered for hours 2 to72.

Though specific dosing regimens are disclosed herein below, it isrecognized that the invention encompasses any administration protocolthat provides for a two-level progesterone or synthetic progestin dosingregimen that provides for initial exposure to higher hourly doses ofprogesterone or synthetic progestin, and subsequent exposure to a lowerhourly doses of progesterone or synthetic progestin. For example, thefirst progesterone or synthetic progestin dosing regime can beadministered by a single bolus injection, followed by a second timeperiod of progesterone or synthetic progestin IV administration.

In still further embodiments, the total infusion dose of progestrone perhour that is to be administered during the first and second time periodsof the two-level progesterone or synthetic progestin dosing regimen ischosen such that a lower total hourly dose of progesterone or syntheticprogestin is given during the first time period and a higher hourly doseof progesterone or synthetic progestin is given during the second timeperiod.

Area under the curve (AUC) refers to the area under the curve thattracks the serum concentration (nmol/L) of progesterone or syntheticprogestin over a give time following the IV administration of thereference progesterone or synthetic progestin standard. By “referenceprogesterone or synthetic progestin standard” is intended theformulation of progesterone or synthetic progestin that serves as thebasis for determination of the total hourly progesterone or syntheticprogestin dose to be administered to a human subject with a traumaticcentral nervous system injury in accordance with the desired constant ortwo-level progesterone or synthetic progestin dosing regimen to achievethe desired positive effect, i.e., a positive therapeutic response thatis improved with respect to that observed without administration ofprogesterone or synthetic progestin. For the determination of the AUCfor the reference progesterone or synthetic progestin standard, see theExperimental Section, Example 1. Accordingly, the total hourly dose ofprogesterone or synthetic progestin to be administered during theconstant or two-level progesterone or synthetic progestin dosing regimencan therefore allow for a final serum level of progesterone or syntheticprogestin of about 100 ng/ml to about 2000 ng/ml, about 100 ng/ml toabout 1000 ng/ml, about 1100 ng/ml to about 1450 ng/ml, about 100 ng/mlto about 250 ng/ml, about 100 ng/ml to about 275 ng/ml, about 100 ng/mlto about 300 ng/ml, about 100 ng/ml to about 325 ng/ml, about 100 ng/mlto about 350 ng/ml, about 100 ng/ml to about 375 ng/ml, about 100 ng/mlto about 400 ng/ml, about 100 ng/ml to about 425 ng/ml, about 100 ng/mlto about 450 ng/ml, about 125 ng/ml to about 250 ng/ml, about 125 ng/mlto about 275 ng/ml, about 125 ng/ml to about 300 ng/ml, about 125 ng/mlto about 325 ng/ml, about 125 ng/ml to about 350 ng/ml, about 125 ng/mlto about 375 ng/ml, about 125 ng/ml to about 400 ng/ml, about 125 ng/mlto about 425 ng/ml, about 125 ng/ml to about 450 ng/ml, about 150 ng/mlto about 250 ng/ml, about 150 ng/ml to about 275 ng/ml, about 150 ng/mlto about 300 ng/ml, about 150 ng/ml to about 325 ng/ml, about 150 ng/mlto about 350 ng/ml, about 150 ng/ml to about 375 ng/ml, about 150 ng/mlto about 400 ng/ml, about 150 ng/ml to about 425 ng/ml, about 150 ng/mlto about 450 ng/ml, about 175 ng/ml to about 250 ng/ml, about 175 ng/mlto about 275 ng/ml, about 175 ng/ml to about 300 ng/ml, about 175 ng/mlto about 325 ng/ml, about 175 ng/ml to about 350 ng/ml, about 175 ng/mlto about 375 ng/ml, about 175 ng/ml to about 400 ng/ml, about 175 ng/mlto about 425 ng/ml, about 175 ng/ml to about 450 ng/ml, about 200 ng/mlto about 300 ng/ml, about 200 ng/ml to about 325 ng/ml, about 200 ng/mlto about 350 ng/ml, about 200 ng/ml to about 375 ng/ml, about 200 ng/mlto about 400 ng/ml, about 200 ng/ml to about 425 ng/ml, about 200 ng/mlto about 450 ng/ml, about 200 ng/ml to about 500 ng/ml, about 200 ng/mlto about 550 ng/ml, about 300 ng/ml to about 400 ng/ml, about 300 ng/mlto about 450 ng/ml, about 300 ng/ml to about 500 ng/ml, about 300 ng/mlto about 550 ng/ml, about 350 ng/ml to about 450 ng/ml, about 350 ng/mlto about 500 ng/ml, about 350 ng/ml to about 550 ng/ml, about 400 ng/mlto about 550 ng/ml, about 500 ng/ml to about 650 ng/ml, about 600 ng/mlto about 750 ng/ml, about 700 ng/ml to about 850 ng/ml, about 800 ng/mlto about 950 ng/ml, about 900 ng/ml to about 1050 ng/ml, about 1000ng/ml to about 1150 ng/ml, about 1100 ng/ml to about 1250 ng/ml, about1200 ng/ml to about 1350 ng/ml, about 1300 ng/ml to about 1500 ng/m,about 1400 ng/ml to about 1600 ng/m, about 1500 ng/ml to about 1700ng/m, about 1600 ng/ml to about 1800 ng/m, about 1700 ng/ml to about1900 ng/m, or about 1800 ng/ml to about 2000 ng/m.

In specific embodiments, the serum level of progesterone or syntheticprogestin comprises about 100 ng/ml, 125 ng/ml, 150 ng/ml, 175 ng/ml,200 ng/ml, 210 ng/ml, 220 ng/ml, 230 ng/ml, 240 ng/ml, 250 ng/ml, 260ng/ml, 270 ng/ml, 280 ng/ml, 290 ng/ml, 300 ng/ml, 310 ng/ml, 320 ng/ml,330 ng/ml, 340 ng/ml, 350 ng/ml, 360 ng/ml, 370 ng/ml, 380 ng/ml, 390ng/ml, 400 ng/ml, 410 ng/ml, 420 ng/ml, 430 ng/ml, 440 ng/ml, 450 ng/ml,460 ng/ml, 470 ng/ml, 480 ng/ml, 490 ng/ml, 500 ng/ml, 510 ng/ml, 520ng/ml, 530 ng/ml, 540 ng/ml, 550 ng/ml, 560 ng/ml, 570 ng/ml, 580 ng/ml,590 ng/ml, 600 ng/ml, 625 ng/ml, 650 ng/ml, 675 ng/ml, 700 ng/ml, 725ng/ml, 750 ng/ml, 775 ng/ml, 800 ng/ml, 825 ng/ml, 850 ng/ml, 875 ng/ml,900 ng/ml, 925 ng/ml, 950 ng/ml, 975 ng/ml, 1000 ng/ml, 1100 ng/ml, 1200ng/ml, 1300 ng/ml, 1400 ng/ml, 1500 ng/ml, 1600 ng/ml, 1700 ng/ml, 1800ng/ml, 1900 ng/ml, or 2000 ng/ml.

In other embodiments, the serum level of progesterone or syntheticprogestin comprises less than 200 ng/ml, 225 ng/ml, 250 ng/ml, 275ng/ml, 300 ng/ml, 310 ng/ml, 320 ng/ml, 330 ng/ml, 340 ng/ml, 350 ng/ml,360 ng/ml, 370 ng/ml, 380 ng/ml, 390 ng/ml, 400 ng/ml, 410 ng/ml, 420ng/ml, 430 ng/ml, 440 ng/ml, 450 ng/ml, 460 ng/ml, 470 ng/ml, 480 ng/ml,490 ng/ml, 500 ng/ml, 510 ng/ml, 520 ng/ml, 530 ng/ml, 540 ng/ml, 550ng/ml, 560 ng/ml, 570 ng/ml, 580 ng/ml, 590 ng/ml, 600 ng/ml, 625 ng/ml,650 ng/ml, 675 ng/ml, 700 ng/ml, 725 ng/ml, 750 ng/ml, 775 ng/ml, 800ng/ml, 825 ng/ml, 850 ng/ml, 875 ng/ml, 900 ng/ml, 925 ng/ml, 950 ng/ml,975 ng/ml, or 1000 ng/ml.

In other embodiments, the serum level of progesterone or syntheticprogestin comprises at least 100 ng/ml, 125 ng/ml, 150 ng/ml, 175 ng/ml,200 ng/ml, 225 ng/ml, 250 ng/ml, 275 ng/ml, 300 ng/ml, 310 ng/ml, 320ng/ml, 330 ng/ml, 340 ng/ml, 350 ng/ml, 360 ng/ml, 370 ng/ml, 380 ng/ml,390 ng/ml, 400 ng/ml, 410 ng/ml, 420 ng/ml, 430 ng/ml, 440 ng/ml, 450ng/ml, 460 ng/ml, 470 ng/ml, 480 ng/ml, 490 ng/ml, 500 ng/ml, 510 ng/ml,520 ng/ml, 530 ng/ml, 540 ng/ml, 550 ng/ml, 560 ng/ml, 570 ng/ml, 580ng/ml, 590 ng/ml, 600 ng/ml, 625 ng/ml, 650 ng/ml, 675 ng/ml, 700 ng/ml,725 ng/ml, 750 ng/ml, 775 ng/ml, 800 ng/ml, 825 ng/ml, 850 ng/ml, 875ng/ml, 900 ng/ml, 925 ng/ml, 950 ng/ml, or 975 ng/ml.

While not being bound by any mechanism of action, the pharmacokineticsof progesterone in patients with traumatic brain injury aresignificantly different than the pharmacokinetics observed in patientswithout traumatic brain injury. Differences include a higher clearance,longer half life and higher volume of distribution, which result inlower than expected serum levels of progesterone. Thus, in oneembodiment of the present invention, administration of progesterone orsynthetic progestin to subjects in need of therapy (e.g., patients withtraumatic brain injury) results in a final serum level of progesteroneor synthetic progestin that is significantly lower than the serum levelachieved in healthy subjects (e.g., subjects without traumatic braininjury) administered the same dose of progesterone or syntheticprogestin. In one embodiment, the serum level of progesterone orsynthetic progestin after administration to a subject in need of therapyis at least about 10 to about 300 ng/ml lower than the serum levelachieved in healthy subjects, e.g., at least about 25, 50, 75, 100, 125,150, 175, 200, 225, 250, 275, or 300 ng/ml lower. In a furtherembodiment, the lower serum level of progesterone or synthetic progestinis due at least in part to a lower clearance rate in subjects in need oftherapy as compared to healthy subjects.

The methods of the present invention also contemplate embodiments wherea subject undergoing a constant progesterone or synthetic progestintherapy or a two-level progesterone or synthetic progestin dosingregimen is given a time period off from progesterone or syntheticprogestin dosing. For example, when a progesterone or syntheticprogestin dosing regime is performed, the time period off fromprogesterone or synthetic progestin can occur between the conclusion ofthe first time period of the two-level progesterone or syntheticprogestin dosing regimen and the initiation of the second time period ofthe two-level progesterone or synthetic progestin dosing regimen. Forexample, one could contemplate the first time period being administeredin a pre-hospital setting, for instance at the site of the trauma. Thesecond time period could then begin upon arrival at a hospital. In theseembodiments, the two-level progesterone or synthetic progestin dosingregimen is interrupted such that progesterone or synthetic progestindosing is withheld for a period of about 15 minutes, 30 minutes, 1 hr, 2hr, 3 hr, 4 hr, 5 hr, 6 hr or more.

In other embodiments, the constant progesterone or synthetic progestintherapy or the two-level progesterone or synthetic progestin therapycomprises a final time period in which the administration ofprogesterone or synthetic progestin is tapered. By “taperedadministration” is meant an administration protocol which reduces thedose of administration to the patient and thereby produces a gradualreduction and eventual elimination of progesterone or syntheticprogestin, either over a fixed period of time or a time determinedempirically by a physician's assessment based on regular monitoring of atherapeutic response of a subject to a traumatic CNS injury. The periodof the tapered progesterone or synthetic progestin administration can beabout 12, 24, 36, 48 hours or longer. Alternatively, the period of thetapered progesterone or synthetic progestin administration can rangefrom about 1 to 12 hours, about 12 to about 48 hours, or about 24 toabout 36 hours.

The drug taper employed could be a “linear” taper. For example, a “10%”linear taper from 500 mg would go 500, 450, 400, 350, 300, 250, 200,150, 100, 50. Alternatively, an exponential taper could be employedwhich, if the program outlined above is used as an example, theexponential taper would be, e.g., 500, 450, 405, 365, 329, 296, 266,239, etc. Accordingly, about a 5%, 10%, 20%, 30%, or 40% linear orexponential taper could be employed in the methods of the invention. Inaddition, a linear or exponential taper of about 1% to 5%, about 6% to10%, about 11% to 15%, about 16% to 20%, about 21% to 25%, about 26% to30%, about 31% to 35%, about 36% to 40% could be employed.Alternatively, the taper schedule can be determined based on thephysician's assessment of the patient's response to therapy. Additionalmethods of tapered administration can be found, for example, in U.S.Provisional Application 60/729,663, filed Oct. 24, 2005, hereinincorporated by reference in its entirety.

Where a subject undergoing therapy in accordance with the previouslymentioned dosing regimens exhibits a partial response, or a relapsefollowing completion of the first cycle of the therapy, subsequentcourses of progesterone or synthetic progestin therapy may be needed toachieve a partial or complete therapeutic response. Thus, subsequent toa period of time off from a first treatment period, which may havecomprised a constant progesterone or synthetic progestin dosing regimenor a two-level progesterone or synthetic progestin dosing regimen, asubject may receive one or more additional treatment periods comprisingeither constant or two-level progesterone or synthetic progestin dosingregimens. Such a period of time off between treatment periods isreferred to herein as a time period of discontinuance. It is recognizedthat the length of the time period of discontinuance is dependent uponthe degree of subject response (i.e., complete versus partial) achievedwith any prior treatment periods of the progesterone or syntheticprogestin therapy.

These multiple treatment sessions are referred to herein as maintenancecycles, where each maintenance cycle comprises a completed constant ortwo-level progesterone or synthetic progestin dosing regimen. By“completed two-level progesterone or synthetic progestin dosing regimen”is intended the subject has been administered both the first period andthe second period of progesterone or synthetic progestin dosing. Thenecessity for multiple maintenance cycles can be assessed by monitoringthe physiological and behavioral improvement of the patient. Theduration between maintenance cycles can be about 1 hr, 15 hr, 1 day, 2day, 3 day, 4 day, 5 day, 6 day or other such time periods fallingwithin the range of about 1 day to about 14 days.

The term “progesterone” as used herein refers to a member of theprogestin family and comprises a 21 carbon steroid hormone. Progesteroneis also known as D4-pregnene-3,20-dione; δ4-pregnene-3,20-dione; orpregn-4-ene-3,20-dione and it its structure is provided below as formula(I). The progesterone used in the methods of the invention can benaturally occurring or synthetic.

Further encompassed by the methods of the invention are syntheticprogestins. As used herein a “synthetic progestin” is a molecule whosestructure is related to that of progesterone, is synthetically derived,and retains the biologically activity of progesterone (i.e., treats atraumatic CNS injury). Representative synthetic progestin include, butare not limited to, modifications that produce 17a-OH esters (i.e.,17α-hydroxyprogesterone caproate), as well as, modifications thatintroduce 6 a-methyl, 6-Me, 6-ene, and 6-chloro sustituents ontoprogesterone (i.e., medroxyprogesterone acetate, megestrol acetate, andchlomadinone acetate). Table 1 provides further, non-limiting examples,of synthetic progestins.

TABLE 1 Classification of Synthetic Progestins Classification Usualclassification by generation* by structure First Second Third EstranesEthynodiol diacetate — — (with ethinyl estradiol:Demulen) Norethindrone(Micronor) Norethindrone acetate (Aygestin) Gonanes NorgestrelLevonorgestrel Desogestrel (with (Ovrette) (Norplant; with ethinylestradiol: ethinyl estradiol: Desogen) Gestodene† Alesse, Nordette)Norgestimate (with ethinyl estradiol: Ortho-Cyclen, Ortho Tri-Cyclen)Pregnanes Medroxyprogesterone — — acetate (Provera) *The traditionalclassification is based on time since market introduction and not onstructural and physiologic differences or efficacy.

The composition comprising progesterone or synthetic progestin which isemployed in the methods of the invention may further comprise aninorganic or organic, solid or liquid, pharmaceutically acceptablecarrier. The carrier may also contain preservatives, wetting agents,emulsifiers, solubilizing agents, stabilizing agents, buffers, solventsand salts. Compositions may be sterilized and exist as solids,particulants or powders, solutions, suspensions or emulsions. In oneembodiment, the progesterone or synthetic progestin is dissolved inethanol, or any other carrier which allows progesterone or syntheticprogestin to dissolve.

The progesterone or synthetic progestin can be formulated according toknown methods to prepare pharmaceutically useful compositions, such asby admixture with a pharmaceutically acceptable carrier vehicle.Suitable vehicles and their formulation are described, for example, inRemington's Pharmaceutical Sciences (16th ed., Osol, A. (ed.), Mack,Easton Pa. (1980)). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the progesterone, either alone, orwith a suitable amount of carrier vehicle.

The pharmaceutically acceptable carrier of the present invention willvary depending on the method of drug administration. The pharmaceuticalcarrier employed may be, for example, either a solid, liquid, or timerelease. Representative solid carriers are lactose, terra alba, sucorse,talc, geletin, agar, pectin, acacia, magnesium stearate, stearic acid,microcrystallin cellulose, polymer hydrogels, and the like. Typicalliquid carriers include syrup, peanut oil, olive oil, cyclodextrin,intralipid, and the like emulsions. Those skilled in the art arefamiliar with appropriate carriers for each of the commonly utilizedmethods of administration. Furthermore, it is recognized that the totalamount of progesterone or synthetic progestin administered as atherapeutic effective dose will depend on both the pharmaceuticalcomposition being administered (i.e., the carrier being used) and themode of administration.

In one embodiment, the carrier comprises cyclodextrin. For example, theformation can comprise progesterone or synthetic progestin dissolved ina 22.5% 2-hydroxypropyl-β-cyclodextrin (Sigma). See, for example, Gosset al. (2003) Pharm. Biochem. and Behavior 76:231-242, the contents ofwhich is herein incorporated by reference. In yet another embodiment,the carrier comprises intralipid. In one embodiment, Intralipid® 20%(Fresenius Kabi pharmaceuticals, Clayton, N.C.) is employed. Thelipophilic properties of Intralipid® 20% allow up to 4 gm ofprogesterone or synthetic progestin per 1 liter of intralipid to bedissolved into solution.

Administration of the progesterone or synthetic progestin may beperformed by many methods known in the art. The present inventioncomprises all forms of dose administration including, but not limitedto, systemic injection, parenteral administration, intravenous,intraperitoneal, intramuscular, transdermal, buccal, subcutaneous andintracerebroventricular administration. Alternatively, the progesteroneor synthetic progestin may be administered directly into the brain orcerebrospinal fluid by any intracerebroventricular technique including,for example, lateral cerebro ventricular injection, lumbar puncture or asurgically inserted shunt into the cerebro ventricle of a patient.Methods of administering may be by dose or by control release vehicles.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the progesterone or syntheticprogestin. The controlled delivery may be exercised by selectingappropriate macromolecules (for example, polyesters, polyamino acids,polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate). The rate of drug releasemay also be controlled by altering the concentration of suchmacromolecules.

Another possible method for controlling the duration of action comprisesincorporating the therapeutic agents into particles of a polymericsubstance such as polyesters, polyamino acids, hydrogels, poly(lacticacid) or ethylene vinylacetate copolymers. Alternatively, it is possibleto entrap the therapeutic agents in microcapsules prepared, for example,by coacervation techniques or by interfacial polymerization, forexample, by the use of hydroxymethyl cellulose or gelatin-microcapsulesor poly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system, for example, liposomes, albumin, microspheres,microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Suchteachings are disclosed in Remington's Pharmaceutical Sciences (1980).

In further embodiments of the present invention, at least one additionalneuroprotective agent can be combined with the progesterone or syntheticprogestin to enhance neuroprotection following a traumatic CNS injury.Such agents include, for example, compounds that reduce glutamateexcitotoxicity and enhance neuronal regeneration. Such agents may beselected from, but not limited to, the group comprising growth factors.By “growth factor” is meant an extracellular polypeptide-signalingmolecule that stimulates a cell to grow or proliferate. When theprogesterone or synthetic progestin is administered conjointly withother pharmaceutically active agents, (i.e., other neuroprotectiveagents) even less of the progesterone or synthetic progestin may betherapeutically effective.

The progesterone or synthetic progestin may be administered per se or inthe form of a pharmaceutically acceptable salt. When used in medicine,the salts of the progesterone or synthetic progestin should be bothpharmacologically and pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare the free active compound or pharmaceutically acceptable saltsthereof and are not excluded from the scope of this invention. Suchpharmacologically and pharmaceutically acceptable salts can be preparedby reaction of a progesterone or a synthetic progestin with an organicor inorganic acid, using standard methods detailed in the literature.Examples of pharmaceutically acceptable salts are organic acids saltsformed from a physiologically acceptable anion, such as, tosglate,methenesulfurate, acetate, citrate, malonate, tartarate, succinate,benzoate, etc. Inorganic acid salts can be formed from, for example,hydrochloride, sulfate, nitrate, bicarbonate and carbonate salts. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium, or calcium salts of thecarboxylic acid group.

Thus the present invention also provides pharmaceutical formulations orcompositions, both for veterinary and for human medical use, whichcomprise the progesterone or synthetic progestin or a pharmaceuticallyacceptable salt thereof with one or more pharmaceutically acceptablecarriers thereof and optionally any other therapeutic ingredients, suchas other neurotrophic agents. The carrier(s) must be pharmaceuticallyacceptable in the sense of being compatible with the other ingredientsof the formulation and not unduly deleterious to the recipient thereof.

The compositions include those suitable for oral, rectal, topical,nasal, ophthalmic, or parenteral (including intraperitoneal,intravenous, subcutaneous, or intramuscular injection) administration.The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active agent intoassociation with a carrier that constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier, a finely divided solid carrier or both, and then, if necessary,shaping the product into desired formulations.

In one embodiment, micronize progesterone or synthetic progestin isused. The micronization process decreases particle size and enhancesdissolution. Prometrian is one such example of a micronized formulationof progesterone.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,lozenges, and the like, each containing a predetermined amount of theactive agent as a powder or granules; or a suspension in an aqueousliquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, adraught, and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which are optionallymixed with a binder, disintegrant, lubricant, inert diluent, surfaceactive agent or dispersing agent. Molded tablets comprised with asuitable carrier may be made by molding in a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredient(s). Such accessory ingredients mayinclude flavorings, suitable preservatives, an agent to retardcrystallization of the sugar, and an agent to increase the solubility ofany other ingredient, such as polyhydric alcohol, for example, glycerolor sorbitol.

Formulations suitable for parental administration conveniently comprisea sterile aqueous preparation of the active compound, which can beisotonic with the blood of the recipient.

Nasal spray formulations comprise purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, or hydrogenated fats orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the active compound dissolved or suspendedin one or more media such as mineral oil, petroleum, polyhydroxyalcohols or other bases used for topical formulations. The addition ofother accessory ingredients as noted above may be desirable.

Further, the present invention provides liposomal formulations of theprogesterone or synthetic progestin and salts thereof. The technologyfor forming liposomal suspensions is well known in the art. When theprogesterone or synthetic progestin or salt thereof is anaqueous-soluble salt, using conventional liposome technology, the samemay be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the compound or salt, the compound or salt will besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed may be of any conventionalcomposition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced may be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations containing the progesterone or syntheticprogestin or salts thereof, may be lyophilized to produce a lyophilizatewhich may be reconstituted with a pharmaceutically acceptable carrier,such as water, to regenerate a liposomal suspension.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired progesterone or syntheticprogestin or a salt thereof or a plurality of solid particles of thecompound or salt. The desired formulation may be placed in a smallchamber and nebulized. Nebulization may be accomplished by compressedair or by ultrasonic energy to form a plurality of liquid droplets orsolid particles comprising the compounds or salts.

In addition to the aforementioned ingredients, the compositions of theinvention may further include one or more accessory ingredient(s)selected from the group consisting of diluents, buffers, flavoringagents, binders, disintegrants, surface active agents, thickeners,lubricants, preservatives (including antioxidants) and the like.

Behavioral assays can be used to determine the rate and extent ofbehavior recovery in response to the treatment. Improved patient motorskills, spatial learning performance, cognitive function, sensoryperception, speech and/or a decrease in the propensity to seizure mayalso be used to measure the neuroprotective effect. Suchfunctional/behavioral tests used to assess sensorimortor and reflexfunction are described in, for example, Bederson et al. (1986) Stroke17:472-476, DeRyck et al. (1992) Brain Res. 573:44-60, Markgraf et al(1992) Brain Res. 575:238-246, Alexis et al. (1995) Stroke 26:2336-2346;all of which are herein incorporated by reference. Enhancement ofneuronal survival may also be measured using the Scandinavian StrokeScale (SSS) or the Barthl Index.

The treatment of a traumatic brain injury can be monitored by employinga variety of neurological measurements. For example, a partialtherapeutic responses can be monitored by determining if, for example,there is an improvement in the subjects a) maximum daily Glasgow ComaScore; b) duration of coma; 3) daily intracranial pressure—therapeuticintensity levels; 4) extent of cerebral edema/mass effect measured onserial CT scans; and, 5) duration of ventilator support. A briefdescription of each of these assays is provided below.

The Glasgow Coma Score (index GCS) is a reflection of the depth ofimpaired consciousness and is best obtained following initialresuscitation (oxygenation, rehydration and support of blood pressure)but prior to use of sedating drugs, neuromuscular blocking agents, orendotracheal intubation.

The duration of coma will be defined as the number of hours from thetime of injury that the subject is unable to purposefully respond tocommands or mechanical stimulation. For non-intubated subjects, thisequates to a GCS score of >8. For intubated patients, this correlateswith a GCS motor score of ≧5. Duration of coma has been found to bepredictive of functional outcome (Uhler et al. (1994) Neurosurgery34(1): 122-8; Jiang et al (1996) Brain Res 735(1): 101-7; andGonzalez-Vidal et al. (1998) Arch Med Res 29(2): 117-24). Time spent ina coma induced pharmacologically for reasons other than brain injuryshould be subtracted in the final analysis.

The intracranial pressure (ICP) of patients with severe TBI is oftenmonitored with an intracranial pressure device. Monitoring ICP canprovide a measure of cerebral edema. However, inherent variability andanalysis complexities due to therapeutic interventions intended onlowering the ICP mire using ICP measurements. To adjust for theseinterventions a therapeutic intensity scale was developed. This scale,known as the Therapeutic Intensity Level (TIL), measures treatmentaggressiveness for elevated ICPs (Allolio et al. (1995) European Journalof Endocrinology 133(6): 696-700; Adashi et al. (1996) Reproductiveendocrinology, surgery, and technology Philadelphia: Lippincott-Raven;and, Beers et al. eds. (1999) The Merck manual of diagnosis and therapy.17th ed., Merck Sharp & Dohme Research Laboratories, Rahway, N.J.).

The extent of cerebral edema and mass effect can be determined by CTscans. For example, the volume of focal lesions can be measured. Masslesions, either high-density or mixed-density abnormalities, will beevaluated by measuring the area of the abnormality as a region ofinterest, multiplying the area by the slice thickness, and summing thesevolumes for contiguous slices showing the same lesion. Each lesion willbe measured three times, and the mean volume will be entered. Thistechnique has been shown to be reliable (Garcia-Estrada et al. (1993)Brain Res 628(1-2): 271-8).

Intracerebral lesions can be further characterized by location (frontal,temporal, parietal, occipital, basal ganglia, or any combination). Whenan edematous zone is present, its volume (the hypodense perimeter) canbe measured and analyzed separately. Midline shift will be measuredusing the septum pellucidum as the midline structure. Theventricle-brain ratio (VBR) will be calculated to quantify the degree ofcerebral atrophy. Levin et al. ((1981) Archives of Neurology38(10):623-9) found that the VBR had satisfactory reliability acrossdifferent examiners, and was related both to the severity of acuteinjury and neurobehavioral sequelae (Hoffman et al. (1994) J Neurotrauma11(4): 417-31).

The duration of ventilator support will be defined as the number ofhours the patient receives positive pressure mechanical ventilation(Uhler et al. (1994) Neurosurgery 34(1): 122-8; Jiang et al. (1996)Brain Res 735(1): 101-7; and Gonzalez-Vidal et al. (1998) Arch Med Res29(2): 117-24). Time spent under ventilator support for reasons otherthan brain injury will be subtracted in the final analysis.

In addition to the neurological measurements discussed above, a partialtherapeutic response can also be assayed through various functional andneuropsychological outcomes. Several standardized measures ofneuropsychological and functional performance are known. For instancesubjects may display an improvement in the Glasgow Outcome Scale(GOS)/Glasgow Outcome Scale Extender (GOSE) and/or in the DisabilityRating Scale (DRS). The Glasgow Outcome Score is one of the most widelyused measures of brain injury recovery in the world (Garcia-Estrada etal. (1999) Int J Dev Neurosci 17(2): p. 145-51). Patients are classifiedinto one of five categories: death, persistent vegetative state, severedisability, moderate disability, and good recovery. It is easy toadminister and score, and has a high degree of reliability and validity.

The Disability Rating Scale (DRS) offers more precision than the GOS formeasuring outcomes of moderate brain injury (Goodman et al. (1996) JNeurochem 66(5): 1836-44). The DRS consists of an eight-item rating ofarousal and awareness, daily living activities, physical dependence, andemployability (Vedder et al. (1999) J Neurochem 72(6):2531-8).Inter-rater reliability for the entire DRS is high (0.97 to 0.98).

The Functional Independence Measure (FIM) can be used to assess physicaland cognitive disability. It contains 18 items in the following domains:self-care, sphincter control, mobility, locomotion, communication, andsocial cognition (Baulieu (1997) Mult Scler 3(2): 105-12). The FIM hasdemonstrated reliability and validity as an outcome measure followingmoderate and severe TBI (Jung-Testas et al. (1994) J Steroid Biochem MolBiol 48(1): 145-54).

The Sickness Impact Profile is one method for measuring self-perceivedhealth status (Schumacher et al. (1995) Ciba Found Symp 191: p. 90-112and Koenig et al. (1995) Science 268(5216):1500-3). It consists of 136questions divided into 12 categories: sleep and rest, eating, work, homemanagement, recreation and pastimes, ambulation, mobility, body care andmovement, social interaction, alertness, behavior, emotional behavior,and communication. It has been widely used across a variety of diseasesand injuries, including head injury (Thomas et al. (1999) Spine24:2134-8). Baseline SIP scores will reflect pre-injury health status,while follow-up scores will examine post-injury functioning.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples which are includedherein for purposes of illustration only, and are not intended to belimiting of the invention, unless specified.

EXPERIMENTAL Example 1

As a first step in assessing the applicability of progesterone therapyin humans, we examined the effects of acute TBI and extracranial traumaon the pharmacokinetics of PG given by intravenous infusion. Multipleblood samples were obtained from 11 female and 21 male trauma patientsreceiving PG and 1 female and 3 male patients receiving placeboinfusions for 72 h. Values for C_(SS), CL, t_(1/2) and V_(d) wereobtained using AUC₍₀₋₇₂₎ and post-infusion blood samples. C_(SS) valueswere 337±135 ng/mL, which were significantly and unexpectedly lower thanthe target concentration of 450±100 ng/mL. The lower C_(SS) isattributed to the CL, which was higher than anticipated. In addition,t_(1/2) was longer and V_(d) was higher than anticipated. There were nosignificant gender differences in any of these parameters. These changesare consistent with the hyperkinetic changes associated with criticalinjury. Our results demonstrate that stable PG concentrations can berapidly achieved following TBI.

Methods Patient Selection:

This study was approved by the Institutional Review Board of EmoryUniversity, the Drug Safety Monitoring Board (NINDS) and the FDA (IND#58,986). After obtaining informed consent, thirty-six patients meetingthe inclusion criteria outlined as follows were studied. Treatments wererandomized using a 4:1 progesterone:placebo ratio. Inclusion criteriarequired that each patient be ≧18 years old, have a closed head injuryarising from blunt trauma, have a moderate to severe brain injury (IndexGlasgow Coma Score (GCS) 4-12) and arrive in the Emergency Departmentand obtain informed consent (from next-of-kin) in less than 11 hourspost injury. Exclusion criteria included: non-survivable injury; noneurological activity (GCS 3); mild TBI (Index GCS 13-15); unknown timeof injury; severe intoxication (ETOH≧250 mg %); spinal cord injury withneuro-deficits; cardiopulmonary arrest; status epilepticus on arrival;blood pressure <90 systolic—on arrival or for ≧5 minutes in durationprior to enrollment; hypoxia on arrival pO₂<60—on arrival or for ≧5minutes in duration prior to enrollment; females who were pregnant;active breast or reproductive organ cancers; or known allergy toprogesterone, or Intralipid® components (egg yolk or soy oil).

Drug Preparation:

Solutions of study drug were prepared by the Investigational DrugService of Emory Healthcare as follows: Progesterone was dissolved in95% ethanol and filtered into sterile vials using a 0.2μ filter.Aliquots of each solution were assayed for final concentration andsterility. Stock solutions of progesterone/placebo were packaged in kits(A, B, C, D or E) that matched the randomized treatment assignments.Each kit contained six vials. Vial 1 contained 15 ml of progesterone orplacebo which was used to prepare the initial bolus and first infusiondose. The remaining 5 vials contained 12 ml of progesterone or placebofor the remaining infusions. Since progesterone is soluble only inalcohol, the diluent used to compound the progesterone solution was 95%ethanol. The placebo kits were also formulated with 95% ethanol. Becauseof the alcohol concentration, doses of study drug were mixed withIntralipid® immediately prior to infusion. Each infusion dose wasadministered over 12 hours and repeated every 12 hours for a total of 72hours. After randomizing a patient, a dosing worksheet based on bodyweight and final progesterone concentration was used to determine thevolume of vial #1 required to be diluted in Intralipid® for a standardloading infusion rate (14 cc/hr) and the first 11 hr of the maintenanceinfusion (10 cc/hr). The dosing worksheet was also used to calculate thevolume of study medication to be diluted in Intralipid® for each of theremaining infusion reservoirs at a standard rate of 10 cc/hr.

Stability of Progesterone Solutions.

For all stability testing, the method of Segall, et al. was used withminor modifications (Segall, et al. (1999) Journal of Pharmaceutical &Biomedical Analysis, 19(5):803-8). The method was originally validatedto assess the stability of medroxyprogesterone acetate and estradiolvalerate tablets. It is an isocratic HPLC-UV method utilizing externalstandardization. A 5 micron, 4.6×250 mm BDS-Hypersil C-18 column(Keystone Scientific) was used and the analyses were completed on anAgilent 1100 model HPLC system with photodiode array detector. Themobile phase consisted of 40% 0.07M ammonium acetate buffer, pH 7.2 and60% acetonitrile. Detection was at 247 nM. A check of system suitabilityyielded 2769 plates per meter (minimum requirement >2500) based on theprogesterone peak and a relative standard deviation (RSD) of 0.80%(minimum requirement 1.0% or less). The tailing factor for theprogesterone peak was 0.5. Reproducibility as assessed by 10 injectionsof the same preparation on multiple different days was always less that10%.

For each assay, progesterone preparations were diluted 1 to 10 withethanol and 1 μL of this dilution was injected. Under these conditions,progesterone eluted at roughly 3.5 minutes. A five point standard curvewas run with each analysis.

Drug Administration:

The progesterone study drug solution was infused at the loading rate of14 mLs/hr (0.71 mg/kg/h) for one hour, followed by a decrease ininfusion rate to 10 mLs/hr (0.5 mg/kg/h) for the remaining 71 hours.Although Intralipid solutions containing progesterone were found to bestable for a minimum of 24 hours, reservoirs of study drug were preparedand changed every 12 hr during the infusion period to minimize the riskof biological contamination. Any interruptions in drug administration orother deviations from the protocol were noted on a drug administrationflow sheet and taken into account when calculating the total number ofmg of progesterone actually administered to each patient.

Sampling Paradigm:

Nine (5 ml) samples were obtained at the following times during theinfusion: pre-infusion (0), 4, 6, 12, 24, 36, 48, 60, and 72 hours. Anadditional five samples were obtained following cessation of infusionat: 0.5, 1, 2, 4 and 8 hours. Samples were allowed to clot, and thencentrifuged. After that, the serum was removed and stored at −70° C.until analyzed.

Serum Progesterone Analysis:

Serum progesterone concentrations were measured using the Immulite®progesterone chemi-luminescent enzyme immunoassay by the ImmunologyLaboratory of the Department of Pathology, Emory University Hospital.The within and between day coefficients of variation for the assay wereboth <10%. We confirmed the accuracy of our assay by comparing theresults of 9 samples over the range 0.5 to 700 ng/mL assayed in ourlaboratory with those assayed by a reference laboratory (The NicholsInstitute, San Juan Capistrano, Calif.).

Pharmacokinetic Analysis:

The primary pharmacokinetic parameter, CL, was estimated as the ratio ofthe dose to area under the serum concentration-time curve (AUC). AUC'swere calculated using the linear trapezoidal rule (Veng-Pedersen (1989)Clin Pharmacokinet, 17(6):424-40). The elimination phase rate constant,k_(e), was calculated from the serum concentration-time data followingthe termination of the infusion using iterative non-linear regression(WinNonlin®, Pharsight Corporation, Mountain View, Calif.). Volume ofDistribution was estimated as the ratio of CL and k_(e). C_(ss) wasestimated as the ratio of Dose and CL. Actual C_(ss) was defined as theconcentration achieved when the slope of the serum concentration-timecurve for three or more consecutive samples was not different from zero.

Statistical Analysis:

A ‘t’ test for repeated measures and a Spearman's rank correlationcoefficient were used to compare the progesterone concentrationsmeasured by our laboratory with those measured by the Nichols Institute.Predicted C_(SS) concentrations were calculated as the ratio of theinfusion rate/clearance. Differences between predicted and measuredC_(SS) were made using a ‘t’ test for repeated measures. A Bland-Altmananalysis was conducted to assess the magnitude of any bias associatedwith this approach (Bland and Altman (1986) Lancet, 1(8476):307-10).Pharmacokinetic parameter comparisons between male and female wereaccomplished using a ‘t’ test for independent means. A p value of lessthan 0.05 was considered the minimum level for rejection of the nullhypothesis.

Results

Thirty-six patients were studied. Thirty-two (21 males and 11 females)received progesterone and four (3 males and 1 female) received a placeboinfusion. There were no significant differences in the pre-infusionprogesterone concentrations between females (2.86±1.37 ng/mL) and males(2.53±1.73 ng/mL)(p<0.5). Pre-infusion progesterone concentrations forthe patients receiving placebo were 2.1±0.8 ng/mL and were notsignificantly different from patients who received progesterone. Inaddition, these pre-infusion values did not significantly change overthe 84 hour time course of the study. FIG. 1 is a representative serumconcentration-time profile for one patient receiving progesterone andone patient receiving a placebo infusion and in whom a complete samplingparadigm was possible. Progesterone concentrations for patientsreceiving a placebo infusion remained constant throughout the studyperiod. C_(SS) concentrations in patients receiving progesterone wererapidly reached and, once achieved, were stable throughout the infusionperiod. Complete peri- and post-infusion sampling was only possible in7/11 females and 10/21 males because of the critical nature of theinjuries sustained by the study patients. C_(SS) values in the currentstudy were lower than expected based on those reported for infusions ofprogesterone in cancer patients (Christen et al. (1993) Journal ofClinical Oncology 11(12):2417-2426).

Table 2 is a summary of the demographic and primary pharmacokinetic datastratified by sex. There were no significant differences between malesand females with respect to any of the parameters in Table 2 with theexception of body weight. As one might expect, the mean body weight forthe males (81.5±16.2 kg) was significantly greater (p<0.003) than thatfor the females (63.9±11.0 kg). Clearance (CL) values were calculatedfrom the total dose of progesterone infused and the AUC_((0-72h)) ratherthan from AUC_((0-∞)) because complete post-infusion blood sampling wasnot possible in a number of the patients for medical reasons. The meanvalue for CL was found to be 1.73±0.72 L/kg/h and was not different inmales (1.66±0.67 L/kg/h) and females (1.88±0.81 L/kg/h). Although adirect comparison is not possible because we did not record heights inour patients and therefore could not calculate body surface areas, CLvalues in the current study are higher than expected from those reportedfor progesterone infusions in cancer patients (Christen et al. (1993)Journal of Clinical Oncology 11(12):2417-2426). Using the value forprogesterone CL from the current serum concentration-time data did notresult in any statistically significant differences between the C_(ss)values predicted by R_(o)/CL (332±121 ng/mL) and those actually measured(337±135 ng/mL) and were not different for either males or females. FIG.2 is a summary of measured and predicted C_(ss) values plotted againstthe line of identity. The Spearman Rank correlation coefficient for thisrelationship was 0.946 (p<0.001). The significance of the relationshipwas confirmed using a Bland-Altman analysis which revealed no systematicbias between the measured and predicted C_(ss) values. The relativedifference between predicted and measured C_(ss) was −0.8±12.2%(mean±SD) (See FIG. 3). FIG. 4 is a plot of measured C_(ss) for eachpatient showing these concentrations were systematically lower than thetarget concentration range predicted from previous studies (Christen etal. (1993) Journal of Clinical Oncology 11(12):2417-2426; Allolio et al.(1995) European Journal of Endocrinology 133(6):696-700). These datasuggest that in trauma patients with moderate to severely head injuriesthe resulting hyperkinetic physiologic state results in a clinicallysignificant increase in progesterone clearance. These data suggest thatto achieve our target concentration of 450±100 ng/mL, the maintenanceinfusion rate should be increased from 0.5 mg/kg/h to approximately 0.8mg/kg/h.

The mean value for terminal half-life was found to be 1.78±1.0 h. Onceagain, there were no differences between males (1.60±0.95 h) and females(2.03±1.08 h) (p<0.4). These values are somewhat longer than thosereported in cancer patient (Christen et al. (1993) Journal of ClinicalOncology 11(12):2417-2426). Volumes of distribution (V_(d)) in thecurrent study are higher than expected from previous reports because ofthe elevation in CL and decrease in terminal elimination phase rateconstant. Although values for males tended to be lower, V_(d)'s were notsignificantly different for males (3.76±2.14 L/kg) and females(5.76±4.21 L/kg) (p<0.22).

TABLE 2 Individual progesterone pharmacokinetic parameters in TBIpatients. Css Css Corrected BW CL Vd Measured Predicted Ro Patient SexAge (kg) GCS (L/kg/h) t_(1/2)(h) (L/kg) ng/mL ng/mL mg/kg/h 1 F 25 72 71.04 2.70 4.04 423 482 0.47 2 F 29 65 8 2.11 2.56 7.78 234 240 0.95 3 F20 57 11 3.60 166 139 1.62 4 F 22 50 6 1.68 0.92 2.24 249 297 0.76 5 F48 73 11 1.20 472 416 0.54 6 F 21 70 4 1.58 1.20 2.73 331 317 0.71 7 F19 85 7 1.16 457 432 0.52 8 F 53 50 7 2.95 2.43 10.35 195 170 1.33 9 F20 55 7 2.25 3.64 11.83 240 222 1.01 10 F 54 57 4 1.91 296 262 0.86 11 F24 69 6 1.19 0.77 1.33 415 420 0.54 Mean ± 30 63.9 7* 1.88 2.03 5.76 316309 0.85 SD 14 11.0 4-11** 0.81 1.08 4.21 110 115 0.37 1 M 52 75 6 1.371.14 2.25 412 366 0.61 2 M 24 70 8 3.84 0.73 4.05 123 130 1.73 3 M 47 9312 0.93 563 538 0.42 4 M 25 70 11 1.55 345 323 0.70 5 M 23 80 6 0.983.30 4.67 499 510 0.44 6 M 29 70 12 1.07 1.77 2.73 368 467 0.48 7 M 2065 10 2.10 2.78 8.43 238 238 0.95 8 M 18 59 6 2.04 1.70 5.01 248 2450.92 9 M 62 66 6 1.33 2.20 4.22 400 376 0.60 10 M 76 84 7 1.55 332 3220.70 11 M 33 100 4 2.33 225 215 1.05 12 M 25 87.7 6 1.36 268 368 0.61 13M 43 112.6 8 1.81 1.44 3.77 225 276 0.82 14 M 18 73 5 1.24 398 402 0.5615 M 46 84 11 1.22 441 410 0.55 16 M 42 75 7 1.87 0.60 1.62 260 268 0.8417 M 34 122 9 1.54 0.37 0.82 332 324 0.69 18 M 42 70 8 1.77 303 283 0.8019 M 65 100 4 0.76 800 662 0.34 20 M 33 75 12 2.41 178 207 1.09 21 M 4280 7 1.71 335 292 0.77 Mean ± 38 81.5^(#) 7* 1.66 1.60 3.76 347 344 0.75SD 16 16.2 4-12** 0.67 0.95 2.14 148 125 0.30 *Median; **Range; ^(#)p <0.003 between males and females.

Discussion

Clinicians have long sought an effective neuroprotective agent to giveto patients shortly following a traumatic brain injury. Thepathophysiology of brain injury is well understood, but researchers havenot identified a drug that can reliably modulate the pathophysiologiccascade of deleterious effects that lead to cellular necrosis, cerebraledema, and consequently, rising intracranial pressure (Chesnut, et al.(1993) Journal of Trauma-Injury Infection & Critical Care, 34(2):216-22;Povlishock and Jenkins (1995) Brain Pathology, 5(4):415-26). Thetreatment of traumatic brain injury is predominantly supportive innature, and revolves around efforts to maintain cerebral perfusionpressure and adequate oxygenation (Brain Trauma Foundation (1996)“Guidelines for the Management of Severe Head Injury,” Journal ofNeurotrauma, 13(11):643-5; Brain Trauma Foundation B (2000) “Managementand Prognosis of Severe Traumatic Brain Injury, Parts I & II,” Journalof Neurotrauma, 17 (June/July):449-627).

A substantial and rapidly growing body of data indicates that thehormone progesterone, a neurosteroid that is naturally found in thebrains of men and women, has potent neuroprotective properties. The datapresented herein obtained during the first pilot, randomized controlledclinical trial of progesterone for treatment of moderate to severe acutetraumatic brain injury (TBI). In addition to testing whether the drug issafe and efficacious for this condition, we sought to determine thepharmacokinetic properties of intravenous progesterone in multi-systemtrauma patients.

The major findings of our investigation are: 1) A solution ofprogesterone in 95% ethyl alcohol is stable for up to 2 years at roomtemperature; 2) Intralipid® solutions containing progesterone in 95%ethyl alcohol are stable for a minimum of 24 hours; 3) A C_(SS) ofprogesterone can be rapidly achieved and maintained in acute, criticallyill traumatic brain injured patients with multi-system trauma using atwo phase intravenous infusion paradigm; 4) Progesterone C_(SS) valuescan be accurately predicted from AUC data; 5) The hyperkineticphysiologic alterations accompanying acute traumatic brain injury resultin significant elevations in CL, t_(1/2), and V_(d) for progesterone; 6)Acute traumatic brain injury, per se, does not result in endogenousrelease of progesterone; and 7) Alterations in progesteronepharmacokinetics following acute traumatic brain injury are not genderdependent. One of the most important goals in clinical pharmacokineticsis obtaining patient specific estimates of the appropriatepharmacokinetic parameters. The use of model independent methods (AUC)is extremely robust for determining patient specific CL. CL is theprimary parameter of interest when drugs are being administered bycontinuous intravenous infusion, since the resultant patient-specificC_(SS) is dependent only on infusion rate and CL. The current studydemonstrates that stable C_(SS) values of progesterone were rapidlyachieved with intravenous administration, making dosing adjustments torealize a target concentration practical in a population of criticallyinjured patients regardless of gender. While the number of patients inthis investigation receiving a placebo infusion is small, repeatedsampling and analysis shows that the initial progesterone concentrationsare constant over the 84-hour time course of study. These data suggestthat endogenous secretion of progesterone is not significantlystimulated by traumatic brain injury, per se. The ultimate goal, ofcourse, is to define the C_(SS) that correlates with optimum treatmentefficacy. Once the pharmacodynamic relationship between steady stateserum concentration of progesterone and clinical outcome is elucidated,the parameters determined in our study may be used to draft an infusionparadigm that optimizes the odds of survival and functional recovery.Since the C_(SS) are rapidly achieved and are stable, patient-specificadjustments in infusion rate to maintain a target concentration shouldbe possible with minimal early blood sampling. If such a pharmacologicintervention proves efficacious, our stability data demonstrate thatstock solutions of progesterone in ethanol, which are tedious toprepare, can be safely used for up to two years. This would allowneurotrauma units immediate access to progesterone solutions andfacilitate rapid treatment implementation.

In 1993, the Brain Injury Foundation convened an international taskforce to develop evidence-based guidelines for treatment of traumaticbrain injury (Brain Trauma Foundation (1996) “Guidelines for theManagement of Severe Head Injury,” Journal of Neurotrauma, 13(11):643-5;Brain Trauma Foundation B (2000) “Management and Prognosis of SevereTraumatic Brain Injury, Parts I & II,” Journal of Neurotrauma, 17(June/July):449-627; Roberts, et al. (1998) J Neurol NeurosurgPsychiatry, 65(5):729-33). With the exception of mannitol andbarbiturates, no pharmacological agents were identified that enhancerecovery.

In the current study, additional drugs were co-administered to optimizethe medical management of these critically injured patients. The drugcombinations and dosing regimens were individualized on apatient-specific basis. As such, there was not a consistent group ofthese drugs given to all patients. While a number of the additional drugclasses, in particular, the anticonvulsants and barbiturates can resultin altered physiology including increases in hepatic blood flow andincreases in oxidative metabolism, we cannot unequivocally determinewhether the increased values for progesterone clearance are a result ofconcomitant drug administration, or traumatic brain injury. Finally,because the drug is available in generic forms it is inexpensive.

Using the results from this study coupled with future findings from adose response efficacy trial, investigators will be able to adjustinfusion rates of progesterone to achieve optimal steady-stateconcentrations. If intravenous infusion of progesterone proves toproduce benefits in acutely brain-injured humans it will represent amajor advance in the treatment of this common and devastating condition.

Example 2

A pilot phase II, randomized, double-blind, controlled trial ofprogesterone for the treatment of a traumatic brain injury waspreformed. The administration protocol was carried out was describedabove in Example 1.

To determine if a therapeutic response was achieved, the followingendpoints were monitored:

-   -   ICP reduction determined by calculating “therapeutic intensity        level” (ICP-TIL);    -   duration of coma (injury to awaking);    -   mortality one-month post injury;    -   neurological outcome 1 month and 1 year post-injury, as        determined by Glasgow outcome scale (GOS), Disability rating        scale (DRS) and Galveston orientation and amnesia test (GOAT).

The preliminary evaluations are as follows. One hundred patients havingmoderate to severe TBI were enrolled in the study, which had arandomized block design 4:1 enrollment. Three days IV administration ofprogesterone [450+/−nmol/L] in both males and females. Theadministration protocol and pharmaceutical composition administered aredescribed in detail in Example 1. Follow up regarding condition occurredat 30 days and 1 year.

Control subjects has a 30.4% mortality rate, while subjects having theprogesterone treatment had a 12.9% mortality rate. The progesteronetreatment group also had a 60% reduction in brain deaths. Table 3summarizes the results.

TABLE 3 Death Frequency percent Treatment row Pct Col Pct A B TotalMedicinal death 5 2 7 5.05 2.02 7.07 Brain death 4 5 9 4.04 5.05 9.09Not Dead 67 16 83 67.68 16.16 83.84 Total 76 23 99 100.00

For test of significance in Table 3, the Ω² test with 2 df wassignificant (p=0.0471). When the treatment groups were compared withrespect to the proportion of subjects experiencing brain death (vs.those who experience medical death of who are not dead), we find thatthe group A has a significantly lower proportion than the B group(p=0.0295 by Fisher's exact test). When the treatment groups withrespect to proportion of subjects experiencing medical death (vs. thosewho experience brain death or who are not dead), it was found that thegroups are not statistically significant (p=0.6622 by Fisher's exactscore).

Example 3

We conducted a clinical trial to assess the safety of progesterone as atreatment for acute TBI. This phase II, randomized, double blind,placebo-controlled clinical trial was conducted at an urban, level Itrauma center. 100 adults presenting within 11 hours of a blunt TBI witha Glasgow Coma Scale score of 4-12 were enrolled with proxy consent.Subjects were randomized on a 4:1 basis to progesterone versus placebo.Blinded observers closely monitored patients for the occurrence ofadverse events, and initial functional outcomes were assessed 30 dayspost-injury. The primary safety outcome was difference in adverse eventrates, including mortality. The primary measure of activity wasdichotomized Glasgow outcome scale extended (GOSE) 30 days post injury.Seventy-seven patients received progesterone; 23 received placebo. Thegroups had very similar demographic and clinical characteristics. Withthe exception of mortality, the rate of adverse events was similar inboth groups. Laboratory values and physiological parameters were similaras well. No serious adverse events were attributed to progesterone. GOSEand other measures of neurological outcome were not significantlydifferent between groups, but progesterone-treated subjects had a lowerall-cause 30 day mortality rate than controls (rate ratio 0.43; 95%confidence interval 0.18-0.99). In this pilot study progesterone causedno observable harms and showed promising signs of activity for treatingTBI.

Introduction

Between 1.5 to 2 million Americans sustain a TBI each year. In the U.S.alone, TBI is annually responsible for 50,000 deaths, 235,000hospitalizations, and 80,000 cases of long term disability.Approximately 37,000 of these victims experience moderate disabilities(Thurman (2001) “The epidemiology and economics of head trauma,” in HeadTrauma: Basic, Preclinical, and Clinical Directions, ed. Miller (Wileyand Sons); Kraus (1997) “Epidemiology of head injury,” in Head Injury,ed. Cooper (2nd ed, Williams & Wilkins Co., Baltimore); Selecki et al.(1982) Australian & New Zealand Journal of Surgery 52(1):93-102; Klauberet al. (1981) Am J Epidemiol 113(5):500-509; Max et al. (1991) Journalof Head Trauma Rehabilitation 6(2):76-91; Gentleman et al. (1992) Injury23(7):471-474; Jones et al. (1994) Journal of NeurosurgicalAnesthesiology 6(1):4-14; Cohadon et al. (1991) Journal of theNeurological Sciences 103 Suppl:S27-31; and, Sakata et al. (1991) BrainInjury 4:411-419.) and 17,000 require specialized care for life. The CDCestimates that 5.3 million Americans are living with disability fromTBI. Lifetime costs of TBI are estimated to exceed $56 billion per year(Thurman (2001) “The epidemiology and economics of head trauma,” in HeadTrauma: Basic, Preclinical, and Clinical Directions, ed. Miller (Wileyand Sons)). We conducted a pilot clinical trial to assess the safety andpotential efficacy of administering intravenous progesterone to victimsof moderate to severe acute traumatic brain injury.

Methods

Study Design The primary objective of this phase II randomized, doubleblind, placebo-controlled trial was to assess potential harms ofadministering intravenous progesterone to acutely brain-injured patientsof both sexes. We also hoped to detect signals of activity.

In the US, IV progesterone had been authorized for experimental use inonly three previous clinical studies, none of which were related to TBI(Aebi et al. (1999) Cancer Chemotherapy & Pharmacology 44(3):259-265;Allolio et al. (1995) European Journal of Endocrinology 133(6):696-700;and, Christen et al. (1993) Journal of Clinical Oncology11(12):2417-2426). The present study shows that IV administration ofprogesterone following TBI would not result in an increased rate ofadverse or serious adverse events.

According to the U.S. Food and Drug Administration, an “adverse event”is any undesirable medical event occurring to a subject in a clinicaltrial, whether or not related to the study drug. This includes eventsnot seen at baseline, or worsened if present at baseline. “Seriousadverse events” are defined as death, immediate risk of death, orsuspicion that use or continued use would result in the patient's death,prolongation of existing hospitalization, persistent or significantdisability/incapacity, or a congenital anomaly/birth defect.

To detect adverse events, blinded observers screened each study subjecton a daily basis to identify a wide range of adverse events, includingbut not limited to those that that could be plausibly related toprogesterone administration. These included any thromboembolic event(deep vein thrombosis, thrombophlebitis, ischemic myocardial infarction,pulmonary embolism, stroke or transient ischemic attack), elevated liverenzymes, temperature elevation, allergic reactions, and hyperglycemia.All laboratory test results obtained during the course of treatment wererecorded and analyzed to detect abnormal levels or worrisome trends. Anindependent internal safety monitor made the determination if an adverseevent was associated with study treatment. Both the FDA and anindependent NIH-appointed data safety monitoring board independentlyreviewed these determinations.

In addition to monitoring subjects for signs of harm, we hoped to detectsignals of activity. We hypothesized that treatment with progesteronemight reduce 30 day mortality and improve a number of short termoutcomes following TBI. For this preliminary study, our primary outcomeof interest was Glasgow Outcome Scale Extended (GOSE) 30 dayspost-injury. Other outcome measures at 30 days included group mortality,Disability Rating Scale score, duration of coma, duration ofpost-traumatic amnesia, and control of increased intracranial pressure.

Setting: The study was conducted at an urban public hospital with over100,000 patients visits per year, the regions only level I trauma centerserving a metropolitan population of more than 4 million.

Selection of participants: Consecutive adult victims of blunt TBI whoreached Grady within 11 hours of injury with a post-stabilization or“index” Glasgow Coma Scale score (iGCS) of 4-12 were eligible forenrollment (FIG. 5). Only 3 potentially eligible patients were missedduring the 2.5-year enrollment period (May 28, 2002 and Sep. 17, 2004).

Whenever a potential candidate was identified, a study investigator cameto the emergency department within 30 minutes to assess eligibility.Exclusion criteria included a blood alcohol concentration of >250 mg/dl;penetrating brain injury; age <18 years; an iGCS of <4 or >12;indeterminate time of injury; pregnancy; cancer; stroke; spinal cordinjury; or unable to secure proxy consent within 11 hours of injury.

Because patients could not consent for themselves, a legally authorizedrepresentative was approached. Proxies were informed of the study'srationale, design, and anticipated benefits and risks. They were assuredthat participation was voluntary, and nonparticipation would not affectthe patient's care. To facilitate comprehension, our consent form wasdrafted at an 8^(th) grade reading level. A Spanish language version wasproduced as well. An investigation new drug authorization to useintravenous progesterone to treat TBI was obtained from the U.S. Foodand Drug Administration, and a NIH-appointed DSMB provided independentguidance and oversight. The hospital's Research Oversight Committee andthe University's Institutional Review Board approved our study. Beforeinitiating enrollment, we briefed leaders of several local advocacyorganizations. We also convened a community advisory board.

Interventions: Following proxy consent, patients were placed in one of 8clinical subgroups defined by gender (male versus female), race (blackversus all others) and TBI severity (moderate versus severe). Withineach subgroup, permuted block randomization was employed to assign 4 outof every 5 consecutive patients to progesterone and the other to placebo(4:1 randomization). This asymmetric approach was adopted at the requestof our NIH-appointed DSMB to maximize the number of patients receivingstudy drug while maintaining blinding.

To insure blinding of all personnel at the hospital, including thehospital pharmacists mixing study infusions, drug kits were preparedoff-site by an Investigational Drug Center. Vials of the study drug andplacebo were identical in appearance and physical properties. To producea set of vials, progesterone was dissolved in 95 percent ethanol andfiltered into sterile vials using a 0.2μ filter. Aliquots were assayedto confirm uniform concentration and sterility. Each study kit containedeither 6 vials of progesterone in ethanol (treatment) or 6 vials ofethanol alone (placebo).

Whenever a patient was enrolled, the next kit in sequence for thatsubgroup was used to prepare infusions. The first vial was mixed inIntralipid™ 20% to deliver a one-hour loading dose of 0.71 mg/kg ofprogesterone at a standard rate of 14 mls/hour, followed by amaintenance infusion of 0.5 mg/kg/h at a standard rate of 10 mls/hour.The remaining vials were used to prepare 5 subsequent 12-hour infusionsat the same standard rate of 10 mls/hour for a total of 3 days oftreatment. Details of drug monitoring are reported elsewhere (Wright etal (2005) J Clin Pharmacol 45(6):640-648).

Clinical services treating brain injured patients at our hospital followa consensus protocol based on the guidelines of the Brain TraumaFoundation (Brain Trauma Foundation B (2000) Journal of Neurotrauma17(June/July):449-627). This protocol governs the treatment of TBIpatients from pre-hospital settings to hospital discharge. A rigorous,stepwise approach is specified to treat episodes of increasedintracranial pressure (ICP). Adopting this protocol assured that withthe exception of treatment group assignment, all study participantsreceived standard treatment for TBI.

Methods of Measurement: To assess drug safety, study personnel roundeddaily to document the occurrence of adverse events (AEs) or seriousadverse events (SAEs). Hourly vital signs (blood pressure, heart rate,respiratory rate, temperature, and pulse oximetry), intracranialpressure readings, and other parameters (mean arterial pressure,cerebral perfusion pressure, and fluid balance), were abstracted fromeach patient's chart. Laboratory values were obtained from thehospital's information system, and concomitant medications andinterventions were noted.

Whenever an SAE occurred, an independent board-certified neurosurgeonassessed its potential relationship to study treatment using pre-definedscale. SAEs were reported within 24 hours to the Institutional ReviewBoard (IRB), our NIH-appointed Data Safety Monitoring Board (DSMB), andthe U.S. Food and Drug Administration. All other adverse events werereported to on a weekly basis.

The infusion was stopped if a patient experienced an anaphylacticreaction, a major thromboembolic event, an unexplained elevation ofserum aspartate aminotransferase (AST) or alanine aminotransferase (ALT)to a level greater than 5,000 IU, or a serum total bilirubin levelgreater than 10 mg/dl. We agreed to prematurely halt enrollment ifeither of 2 interim analyses revealed that one group or the otherexperienced a significantly higher rate of SAEs, including mortality,than the other. These rules were based on O'Brien-Fleming boundaries(O'Brien and Fleming (1979) Biometrics 35(3):549-556), constructed usingan alpha spending approach (DeMets and Lan (1994) Statistics in Medicine13(13-14):1341-1352; discussion 1353-1346).

To determine if the study drug had a beneficial impact on patients, wecollected a variety of physiological and functional measures. Theseincluded: hourly intracranial pressure measurements; duration of coma,defined as the number of hours from injury to awakening (GCS>8 or motorscore >5), and duration of post-traumatic amnesia, defined as the numberof days until a subject achieved two consecutive Galveston Amnesia andOrientation test scores of 75 or better. Thirty days following eachinjury event, we assessed each patient's Glasgow Outcome Score Extended(GOSE) and Disability Rating Scale (DRS). Patients who were severelyimpaired were classified as “not testable”—a surrogate marker for a pooroutcome. Reliability codes were used to record reasons fornon-administration of a particular measure, such as physical impairment(e.g., hemiparesis) cognitive impairment (e.g., could not understandinstructions), or intoxication. One-year outcomes will be reported at alater date.

Data collection and processing: Data collection was guided by a formaldata management plan and standard operating procedures manual. Datacollected at the bedside were recorded on paper case report forms (CRFs)and subsequently double entered into a web-based ORACLE® database.Entered CRFs were not accepted as valid unless the double entriesmatched and all range checks were met. Special edit queries wereconstructed to generate transport files for importing into SAS® foranalysis.

Outcome Measures: The primary aim of our study was to assess the safetyof treatment with progesterone. We hypothesized that treatment andcontrol groups would experience similar rates of SAE's and AE's. Oursecondary aim was to look for signs of drug activity by assessingseveral measures of outcome. Our a priori primary measure of outcome wasthe Glasgow Outcome Scale—Extended (GOSE) (Teasdale et al. (1998)Journal of Neurotrauma 15(8):587-597). Other outcome measuresincluded: 1) death within 30 days of injury 2) duration of coma (Levin(1995) Journal of Neurotrauma 12(5):913-922); 3) duration ofpost-traumatic amnesia (Levin et al. (1979) Journal of Nervous & MentalDisease 167(11):675-684); 4) mean intracranial pressure and intracranialpressure therapeutic intensity level (ICP-TIL) (Maset et al. (1987)Journal of Neurosurgery 67(6):832-840) and 5) the Disability RatingScale (DRS) (Hall et al. (2001) Arch Phys Med Rehabil 82(3):367-374).

Primary Data Analysis—Treatment and placebo groups were compared withrespect to a variety of demographic, historical and prehospitalcharacteristics to ensure that important independent predictors ofoutcome were equally distributed. Next, the groups were compared withrespect to rates of adverse and serious adverse events, using Fisher'sexact test. Generalized linear model analysis using a negative binomialdistribution was used to compare rates of events that occurred multipletimes per patient within the first 30 days (McCullagh and Nelder (1989)Generalized Linear Models (2nd ed, Chapman & Hill)). Then, groupspecific differences in 30-day outcomes. Fisher's exact test was used toanalyze GOSE scores dichotomized into “good or moderate recovery” versusall other levels. Wilcoxon's rank sum test was used to compare groupspecific DRS scores. Mean and median durations of coma andpost-traumatic amnesia were compared using student's t-test. Allanalyses were stratified on an a priori basis by brain injury severity(iGCS 4-8 (severe) versus iGCS 9-12 (moderate)). Longitudinal mixedeffects models were used to analyze ICP-TIL as well as other hourly ordaily clinical measurements from enrollment through treatment day 4.

To insure that any observed differences in mortality were associatedwith the study treatment rather than confounding clinical factors,additional multivariate analyses were performed. Variables determined tobe independently associated with all-cause mortality or CNSrelated-death, such as iGCS (dichotomized into moderate versus severe),injury severity score and Marshall CT score were incorporated in astepwise logistic regression analyses. Because GCS often fluctuatesduring the first few hours after injury, additional stepwise logisticregressions were performed using dichotomized GCS 1 day post-injury.

Results

Screening and Enrollment—A total of 281 patients were screened. Threepotentially eligible patients were missed and 18 patients could not beenrolled because their identity was unknown or a proxy could not becontacted within 11 hours of injury. Six potentially eligible patientswho presented during one of 3 procedural “holds” could not be enrolled.One patient was excluded after consent but prior to randomizationbecause the treating team decided that his injuries were non-survivable.Eleven eligible patients were not enrolled because their proxy declinedto consent. (FIG. 1) Non-participants resembled participants withrespect to gender, race, and mechanism of injury.

Characteristics of study subjects—Seventy-one patients were male; 34were black. Mean age was 36 years. Seventy-two patients (72%) had aniGCS of 4-8; the remainder had a score of 9-12. More than 80% ofinjuries were caused by a motor vehicle crash or a fall. Most patientsreached the hospital within an hour of injury; 58 percent by helicopter.Because it frequently took several hours to locate a representative forproxy consent, mean time from injury to initiation of study infusion was6.3 (95% CT 5.9-6.8) hours in the progesterone group and 6.2 (95% CT5.9-6.6) hours in the placebo group.

Randomization—77 subjects were randomized to progesterone; 23 toplacebo. Treatment groups were highly similar with respect to gender,age, race, iGCS, mechanism of injury, revised trauma score, injuryseverity score, time from injury to E.D. arrival, time to studytreatment, Marshal CT score (Marshall et al. (1991) J Neurosurgery 75(suppl):S14-20), and E.D. disposition (Table 4).

Dosing and protocol compliance—Our pharmacokinetic findings are reportedelsewhere (Wright et al. (2005) J Clin Pharmacol 45(6):640-648). Onepatient randomized to progesterone died before the study drug could beinitiated. She was retained in our analysis under the principle of“intention to treat.” All other members of the treatment group and nomembers of the control group had high serum levels of progesterone intheir sera during drug administration. Minor protocol violations, suchas brief delays in changing I.V. bags, were common. Sufficient solutionwas provided to prevent these from interrupting infusion.

Six major protocol violations occurred. Four involved prolongedinterruptions of the infusion, one involved a dosing error, and oneinvolved inappropriate enrollment of a motor vehicle crash victim. Whena repeat CT scan on the second hospital day revealed an ischemic stroke,his progesterone infusion was promptly stopped. Subsequent review of theadmission CT scan showed subtle but clear signs of the stroke, which wastraced to a traumatic carotid artery dissection. Because the strokepredated treatment, this incident was classified as a major protocolviolation rather than a SAE.

Safety—Aggregate and individual rates of adverse and serious adverseevents were not different between groups (Table 5). This was truewhether AEs and SAEs were analyzed by any occurrence or by mean episodesper subject. Laboratory values of the treatment groups were remarkablysimilar, whether analyzed by group means or the frequency with which aspecified test value exceeded pre-specified thresholds.Progesterone-treated subjects experienced a significantly lower rise inmean temperature over the infusion interval compared to controls. Thiswas determined by analyzing a treatment by time interaction term forprogesterone versus control patients, with the slope=−0.0055 (95% CI,−0.010 to −0.001).

The only adverse events specifically ascribed to administration ofprogesterone were two cases of superficial phlebitis at the IV site.Both resolved spontaneously. Three patients, all of whom receivedprogesterone, developed a deep vein thrombosis between 6 to 23 daysfollowing completion of the infusion. All 3 cases were treated withoutincident. Two patients suffered ischemic strokes. One in a patientrandomized to progesterone, occurred prior to treatment and wasconsidered a major protocol violation. The other involved a patientrandomized to placebo. A patient randomized to progesterone sustained amyocardial infarction two days after the study infusion was completed.At the time, he was receiving high-dose neosynephrine in an effort toboost his cerebral perfusion pressure. Post-mortem revealed nointra-coronary thrombosis.

Signals of benefit—During the first 4 days post-injury, meanintracranial pressure levels (ICPs) of progesterone-treated subjectswith monitors in place remained stable, while mean ICPs amongplacebo-treated subjects with ICP monitors in place tended to rise.However, these trends were not statistically significant. Mean ICP-TILscores did not significantly differ between groups (Table 6).

Severe TBI patients (iGCS 4-8) treated with progesterone remained incoma significantly longer than survivors who received placebo (meanduration 10.1 days (7.7, 12.5) versus 3.9 days (2.5, 5.4) respectively).The mean duration of posttraumatic amnesia did not significantly differbetween groups (Table 7). Ten of 77 patients (13 percent) randomized toprogesterone died within 30 days of injury, compared to 7 of 23 patients(30.4 percent) randomized to placebo (rate ratio 0.43, 95% CI0.18-0.99). When the analysis was restricted to the 99 subjects whoreceived treatment, this difference was more significant (rate ratio0.39, 95% confidence interval 0.16, 0.93). Deaths due to neurologicalcauses tended to be lower in the treatment group than controls (rateratio 0.30, 95% confidence interval 0.08-1.12) while deaths from non CNScauses did not appreciably differ. The association between treatmentgroup and mortality remained robust in multivariate models, includingseveral based on dichotomized GCS at 24 hours (Table 7).

We were able to contact 92 percent of survivors 30 days post-injury toassess their functional status. Our primary outcome measure,dichotomized GOSE, did not significantly differ between groups. The DRSscores of severely brain injured patients were similar as well. However,moderately brain-injured patients randomized to the study drug achievedsignificantly better DRS scores, on average, than those randomized toplacebo (Table 7).

Discussion

Because progesterone has not been previously used to treat acutetraumatic brain injury, we conducted a pilot, phase II study to assesspotential harms. Arriving patients were carefully screened foreligibility. Ninety nine percent of potentially eligible patients werescreened, and 90 percent of those who met inclusion criteria wereenrolled with proxy consent. Treatment and control patients were wellmatched by injury severity, time to treatment and other independentpredictors of outcome.

The decision to secure proxy consent rather than seek exemption frominformed consent delayed initiation of treatment an average of 6.5hours. Although one animal study has suggested that progesterone mayproduce beneficial effects as late as 24 hours post-injury, themagnitude of benefit was greatest when treatment was administered within2 hours of injury (Roof et al. (1996) Exp Neurol 138(2):246-251).

Three members of the treatment group developed deep vein thrombosis—theearliest 6 days post-infusion. This frequency is well within ourinstitution's historical incidence of DVT in major trauma patients(unpublished data). With the exception of mortality, treatment andcontrol groups experienced similar rates of AEs and SAEs. They also hadvery similar lab and physiological values.

Our secondary goal was to detect signs of drug activity. We chose GOSEas our primary outcome measure because it the most widely used standardin the brain injury literature. We observed promising signs of activity.

No differences were found in mean ICP or mean ICP-TILs.

There was no significant difference between treatment groups withrespect to duration of post-traumatic amnesia and 30-day GOSE. However,the 30-day mortality rate among subjects randomized to the treatmentgroup was less than half that of the control group. This differencepersisted after other important predictors of outcome were taken intoconsideration.

Severely brain-injured patients in the treatment group had a longer meanduration of coma than those in the control group. This may represent a“survivor effect”. If progesterone prevented the deaths of severalpatients during the 30 day follow up period, it is not surprising thatthese survivors remained in coma for a longer duration of time. One-yearsurvival and functional outcomes will be reported at a later date.

In retrospect, we would have preferred to enroll patients with exceptionto informed consent. This would have allowed us to start the studytreatment much sooner, and enroll patients who were lost because wecould not find a legally authorized representative within the enrollmentwindow. Earlier administration of the study drug might have producedgreater evidence of activity. We recognized, however, that this is thefirst human trial of progesterone in the setting of acute brain injuryand our study was primarily designed to assess drug safety rather thanactivity. This is why we enrolled patients with proxy consent andaccepted potentially significant treatment delays (up to 11 hours) tomaximize recruitment. Based on our encouraging findings with regards tosafety, we hope to conduct a larger trial under the federal regulatoryframework that allows exception from consent in limited circumstances(Federal regulations of 21CFR50.24). This will enable earlier initiationof treatment and maximize the opportunity to detect any evidence ofneuroprotective effects.

In summary, this study represents an important step in assessing theutility of progesterone for treating acute traumatic brain injury. TBIis a leading cause of death and disability worldwide. No pharmacologicalagent has been shown to improve outcomes. We previously reported thatprogesterone can be accurately administered in intravenous form tovictims of TBI (Wright et al. (2005) J Clin Pharmacol 45(6):640-648).This analysis offers preliminary evidence that this treatment causes noharm and may have disease-modifying activity. A clinical trial involvingmore subjects, 1:1 randomization, and a short enrollment window iswarranted. If it corroborates our findings, this will represent a majoradvance in brain injury care.

TABLE 4 Characteristics by Group: Participants in ProTECT ™ (N = 100)Characteristic Overall Progesterone Placebo p-value* Number of Subjects100 77 23 N/A Mean Age (X ± sd) 35.8 ± 15.0 35.3 ± 14.3 37.4 ± 17.4 0.54Male (%) 71% 71% 70% 0.86 African American (%) 35% 34% 39% 0.64Mechanism of Injury (%) (n = 100) Motor Vehicle 76 74 83 0.58 PedestrianStruck 3 4 0 (mvc vs. all Bicycle 3 3 4 other) Fall 7 6 9 Other 11 13 4Index GCS (% severe) 72% 73% 70% 0.77 24 hr GCS (% severe) 61% 70% 50%0.23 Injury Severity Score (X ± sd) 24.2 ± 9.2  24.5 ± 9.9  23.3 ± 6.4 0.50 Revised Trauma Score (X ± sd) 6.1 ± 0.6 6.1 ± 0.6 6.2 ± 0.7 0.83Probability of Survival (P ± sd) 0.9 ± 0.2 0.9 ± 0.2 0.8 ± 0.1 0.53Initial CT scan Marshall Score⁶⁷ 2.8 ± 1.6 3.0 ± 0.2 2.3 ± 0.3 0.09(1-5) Time injury to arrival (X ± sd) 50.3 ± 30.3 49.5 ± 32.3 54.3 ±32.3 0.42 min Time injury to infusion (X ± sd) 379.2 ± 118.0 380.7 ±125.6 374.0 ± 91.2  0.78 min •p value = progesterone group versusplacebo group

TABLE 5 30-Day Adverse Event Rates by Treatment Group Relative Risk (95%Progesterone (%) Placebo (%) confidence interval) Acute respiratorydistress syndrome 2.6 4.4 0.60 (0.06, 6.29) Central nervous systeminfection 1.3 0.0 — Cardiac Arrhythmia 5.2 17.4 0.30 (0.08, 1.10)Cholestatic Jaundice 6.5 0.0 — Death within 30 days 13.0 30.4 0.43(0.18, 0.99) Fever 70.1 82.6 0.85 (0.67, 1.08) Gastrointestinal Bleed5.2 0.0 — Hyperglycemia-non DM 27.3 30.4 0.90 (0.44, 1.84) Hypertension11.7 8.7 1.34 (0.31, 5.79) Hypotension 9.1 21.7 0.42 (0.15, 1.19)Hypothermia 5.2 8.7 0.60 (0.12, 3.06) Hypoxemia 11.7 13.0 0.90 (0.26,3.04) Increase Liver Enzyme 6.5 4.4 1.49 (0.18, 12.15) Phlebitis atInjection Site 1.3 0.0 — Rash or Hives 2.6 0.0 — Syndrome ofinappropriate ADH 1.3 0.0 — Seizures 5.2 0.0 — Sepsis 2.6 0.0 — Shock2.6 0.0 — Suspected Pneumonia 11.7 4.4 2.69 (0.46, 20.12) Tachycardia24.7 13.0 1.89 (0.61, 5.83) Thromboembolic Disease 3.9 0.0 —

TABLE 6 Physiological Parameters Infusion Day Progesterone Group PlaceboGroup DAY n mean 95% CI n mean 95% CI Intracranial Pressure TherapeuticIntensity Level 0 16 2.6 1.9, 3.4 5 3.8 1.9, 5.7 1 27 2.7 1.4, 4.1 9 2.71.3, 4.1 2 26 3.2 1.9, 4.5 10 4.5 1.2, 7.8 3 17 3.7 1.1, 6.3 9 4.2 0.6,7.9 4 15 2.8 0.6, 5.0 5 6.0   0, 12.3 Intracranial Pressure (mm Hg) 0 1716.0 12.3, 19.7 5 13.13  8.1, 18.2 1 36 17.1 12.6, 21.5 12 14.69 10.1,19.3 2 34 15.4 13.2, 17.5 12 17.32 12.1, 22.6 3 34 16.0 13.8, 18.2 1218.27 13.3, 23.2 4 25 17.7 14.8, 20.7 12 19.95 13.8, 26.1 CerebralPerfusion Pressure (mmHg) 0 13 70.3 61.9, 78.8 3 71.9 48.4, 95.4 1 3673.4 66.2, 80.6 12 76.8 71.5, 82.0 2 34 75.9 71.7, 80.1 12 74.9 70.6,79.1 3 34 74.9 70.7, 79.2 12 75.6 70.8, 80.4 4 25 73.8 68.0, 79.6 1173.2 67.2, 79.1 Systolic Blood Pressure (mmHg) 0 68 129.4 125.6, 133.218 127.6 119.2, 136.0 1 76 130.2 126.5, 133.9 22 129.9 124.0, 135.7 2 75133.5 130.2, 136.8 23 133.0 125.9, 140.1 3 75 133.8 130.1, 137.6 22137.0 130.6, 143.9 4 73 132.7 128.6, 136.9 21 137.8 132.5, 143.4Diastolic Blood Pressure (mmHg) 0 68 69.5 66.6, 72.5 18 66.6 60.3, 72.91 76 67.4 65.1, 69.8 22 66.4 62.6, 70.1 2 75 67.2 64.8, 69.7 23 65.760.7, 70.8 3 75 67.5 65.3, 69.6 22 66.4 62.2, 70.6 4 73 67.3 65.2, 69.421 67.3 63.6, 71.1 Temperature (degrees centigrade) 0 35 37.0 36.6, 37.411 36.9 36.3, 37.6 1 76 37.4 37.3, 37.6 22 37.4 37.1, 37.7 2 75 37.437.3, 37.6 23 37.7 37.4, 38.0 3 75 37.4 37.3, 37.5 22 37.7 37.4, 37.9 473 37.5 37.3, 37.6 21 37.7 37.4, 38.0 Fluid Balance (+ mls) 1 76 767.9312.8, 1223.0 23 834.7    0, 1794.9 2 76 1189.5 645.9, 1733.0 23 1282.2 583.4, 1981.0 3 75 802.0 401.5, 1202.5 22 1292.6  748.1, 1837.0 4 75818.7 386.3, 1251.2 20 812.0  66.9, 1557.2 Parameters ExceedingThreshold Values Percent of Patients with Clinical Values Exceeding theThreshold Progesterone Group Placebo Group n denominator % # n % p-valueMAP < 70 22 76 29.0 10 23 43.5 0.21 CPP < 60 18 37 48.7 5 12 41.7 0.75ICP > 25 12 37 32.4 5 12 41.7 0.73 Systolic BP < 90 22 76 29.0 10 2343.5 0.21 Mean Duration of Pressures Exceeding Threshold Values (hours)Progesterone Group Placebo Group Duration (hrs) n mean std error n meanstd error Wilcoxon MAP < 70 76 2.5 0.7 23 3.4 1.40 0.24 CPP < 60 37 6.92.9 12 2.4 1.18 0.56 ICP > 25 37 5.0 2.5 12 11.3 7.88 0.46 Systolic BP <90 76 2.7 0.7 23 3.5 1.40 0.25 Mean Frequency of Pressures ExceedingThreshold Values Progesterone Group Placebo Group # Rate/1000 #Rate/1000 Consecutive consecutive Consecutive consecutive p- EventOccurrence Readings readings Occurrence Readings readings value MAP < 70128 4334 29.5 0 1477 41.3 0.81 CPP < 60 183 1969 92.9 23 816 28.2 0.41ICP > 25 145 2067 70.2 121 828 146.1 0.61 Systolic 132 4112 32.1 62 136545.4 0.81 BP < 90 MAP = mean arterial pressure, CPP = cerebral perfusionpressure, ICP = intracranial pressure, BP = blood pressure

TABLE 7 Outcomes Variables 30 days Post Injury Progesterone GroupPlacebo Group Total N 77 23 95% Mortality Risk Rate Confidence n % n %Ratio Interval All cause mortality (ITT)* 10 13.0 7 30.4 0.43 0.18, 0.99All cause mortality (TR)# 9 11.8 7 30.4 0.39 0.16, 0.93 Neurologicaldeaths# 4 5.3 4 17.4 Non-neurological deaths# 5 6.6 3 13.0 Survived > 30days# 67 88.2 16 69.6 Total and Dichotomized Glasgow Outcome Score -Extended 95% Risk Rate Confidence Disability level n % % n % % RatioInterval Dead 10 14.2 7 31.8 Vegetative State 5 7.1 0 0 Lower Severe 2840.0 70.0 7 31.8 81.8 Upper Severe 6 8.6 4 18.2 Lower Moderate 8 11.4 418.2 1.65 0.63, 4.29 Upper Moderate 7 10.0 30.0 0 0 18.2 Lower Good 34.3 0 0 Upper Good 3 4.3 0 0 95% 95% Confidence Confidence n MeanInterval n Mean Interval Disability Rating Score Index GCS = 4-8 Employ46 2.7 2.4, 2.9 9 2.4 1.9, 3.0 Function 46 2.9 2.3, 3.5 9 1.8 0.7, 2.8Total DRS 45 10.7  8.0, 13.4 9 4.4 2.8, 6.1 Index GCS = 9-12 Employ 151.8 1.1, 2.5 6 3 — Function 15 1.5 0.5, 2.6 6 3.8 2.6, 5.1 Total DRS 155 1.6, 8.4 6 12.7  7.0, 18.4 Duration of Coma (days) Initial GCS = 4-855 10.11  7.7, 12.5 16 3.9 2.5, 5.4 Initial GCS = 9-12 20 4.1 1.4, 6.8 76.1   0, 13.2 Duration of Post-Traumatic Amnesia (days) Initial GCS =4-8 37 18.6 15.2, 22.0 9 12.8  5.2, 20.4 Initial GCS = 9-12 15 10.7 6.2, 15.3 3 18.3   0, 46.9 *Analyses of intention to treat; #Analysesof treatment received, one patient died prior to receiving study drug

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A method of treating a traumatic brain injury in a human subject inneed thereof, said method comprising administering to said subject atleast one cycle of therapy, wherein said cycle of therapy comprisesadministering a therapeutically effective two-level intravenous dosingregime of progesterone, said two-level dosing regime comprising a firsttime period, wherein a higher hourly infusion dose of progesterone isadministered to the subject, followed by a second time period, wherein alower hourly infusion dose of progesterone is administered to saidsubject.
 2. The method of claim 1, wherein the first time periodcomprises an infusion dose of progesterone of about 0.1 mg/kg/hr toabout 7 mg/kg/hr.
 3. The method of claim 2, wherein the first timeperiod comprises an infusion dose of progesterone of about 0.5 mg/kg/hrto about 1 mg/kg/hr.
 4. The method of claim 3, wherein the first timeperiod comprises an infusion dose of progesterone of about 0.7 mg/kg/hr.5. The method of claim 1, wherein the second time period comprises aninfusion dose of progesterone of about 0.05 mg/kg/hr to about 5mg/kg/hr.
 6. The method of claim 5, wherein the second time periodcomprises an infusion dose of progesterone of about 0.5 mg/kg/hr toabout 1 mg/kg/hr.
 7. The method of claim 6, wherein the second timeperiod comprises an infusion dose of about 0.5 mg/kg/hr.
 8. The methodof claim 1, wherein the second time period comprises about a 24 hour toabout a 120 hour period.
 9. The method of claim 8, wherein the secondtime period comprises about a 71 hour period.
 10. The method of claim 1,wherein the first time period comprises an infusion dose of progesteroneof about 0.7 mg/kg/hr, the second time period comprises an infusion doseof about 0.5 mg/kg/hr, the first time period comprises about 1 hour, andthe second time period comprises about a 71 hour period.
 11. The methodof claim 1, wherein the two-level intravenous dosing regime ofprogesterone results in a progesterone serum level in said subject ofabout 100 ng/ml to about 2000 ng/ml.
 12. The method of claim 11, whereinthe progesterone serum level is about 100 ng/ml to about 1000 ng/ml. 13.The method of claim 12, wherein the progesterone serum level is about200 ng/ml to about 450 ng/ml.
 14. The method of claim 12, wherein theprogesterone serum level is about 350 ng/ml to about 450 ng/ml.
 15. Themethod of claim 1, wherein the two-level intravenous dosing regime ofprogesterone results in a progesterone serum level in said subject ofless than 450 ng/ml.
 16. The method of claim 1, further comprising athird time period, wherein said third time period comprises a taperedadministration of the progesterone to the subject.
 17. The method ofclaim 1, wherein the first and the second time periods are continuous.18. The method of claim 1, wherein the first and the second time periodsare discontinuous.
 19. The method of claim 1, wherein said first timeperiod comprises a bolus injection.
 20. A method for treating atraumatic brain injury in a human subject in need thereof comprisingadministering to said subject a therapeutically effective concentrationof progesterone.
 21. The method of claim 20, wherein said effectiveconcentration of progesterone is administered intravenously.
 22. Themethod of claim 21, wherein said effective concentration of progesteronecomprises a daily dose of 12 mg/kg of body weight.
 23. The method ofclaim 22, wherein said daily dose is administered within 24 hours postinjury.
 24. The method of claim 20, wherein said progesterone is in acyclodextrin carrier.
 25. The method claim 21, further comprisingadministering to said human subject a subsequent daily dose comprisingan effective concentration of progesterone, wherein the effectiveconcentration of progesterone in the subsequent daily dose unit is about12 mg/kg of body weight.
 26. The method of claim 21, said methodcomprising administering to said subject at least one cycle of therapy,wherein said cycle of therapy comprises administering a therapeuticallyeffective constant intravenous dosing regime of progesterone.
 27. Themethod of claim 26, wherein the constant intravenous dosing regimecomprises an infusion dose of progesterone of about 0.1 mg/kg/hr toabout 7 mg/kg/hr.
 28. The method of claim 27, wherein the constantintravenous dosing regime comprises an infusion dose of progesterone ofabout 0.5 mg/kg/hr to about 1 mg/kg/hr.
 29. The method of claim 28,wherein the constant intravenous dosing regime comprises an infusiondose of progesterone of about 0.7 mg/kg/hr.
 30. The method of claim 26,wherein the constant intravenous dosing regime of progesterone resultsin a progesterone serum level in said subject of about 100 ng/ml toabout 2000 ng/ml.
 31. The method of claim 30, wherein the progesteroneserum level is about 100 ng/ml to about 1000 ng/ml.
 32. The method ofclaim 31, wherein the progesterone serum level is about 200 ng/ml toabout 450 ng/ml.
 33. The method of claim 31, wherein the progesteroneserum level is about 350 ng/ml to about 450 ng/ml.
 34. The method ofclaim 26, wherein the constant intravenous dosing regime of progesteroneresults in a progesterone serum level in said subject of less than 450ng/ml.